Reagents and methods for mineralization of tooth enamel

ABSTRACT

Reagents and methods for whitening and remineralizing teeth using biomineralizing peptides are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. No. 62/279,418 filed Jan. 15, 2016 and PCT applicationPCT/US2016/013301 filed Jan. 13, 2016, the contents of which areincorporated herein by reference.

BACKGROUND

Accumulation of various chromogens/discolorants, for example, food andtobacco that come into contact daily onto tooth surfaces and theirsubsequent penetration into deeper regions (dentin), cause toothdiscoloration. Furthermore, the process of aging, disease, trauma,certain medications, certain congenital conditions, and environmentaleffects can also cause teeth to become discolored. Although discoloredteeth do not cause health problems, since bright white teeth are usuallyconsidered to be cosmetically desirable, there is a great deal ofinterest in developing compositions and methods for whitening teeth.

There are several techniques for whitening or bleaching of teeth.Professional whitening methods, also known as “in-clinic” whiteningstrategies, are considered presently as the most effective methods.These in-clinic whitening strategies typically involve the applicationof high-concentration peroxide products (up to 35%) and other abrasivechemicals to the discolored area. These peroxide species penetrate thestained area (up to underlying dentin layer) and dissolve theaccumulated discoloring agents through an oxidation process. To achievedesired effects more quickly, such oxidation reactions on teeth areoften assisted with external laser light application, which heats up thereaction site and thereby increases the rate oxidation reaction.Furthermore, utilization of titanium dioxide up to 10% is oftenpreferred to facilitate these photo-catalytic reactions. Other in-clinicwhitening techniques involve superficially removing the enamel layerwith abrasive instruments or pumice followed by treatment withadditional caustic agents.

There are several drawbacks of these in-clinic techniques. First of all,the abrasive chemicals and peroxide agents causes enameldemineralization and results with teeth sensitivity. In most casessore/injured gums as well as bad taste of the product itself cause muchdiscomfort to the patients. Further, patients are required to make aclinic appointment to get this medical service.

Other existing products for at-home use contain considerably lowerconcentrations of active oxidizing agents and, thus, are generally lesseffective than in-clinic whitening strategies and products. Therefore,dramatic whitening effect can only be achieved by the repeatedapplications of these reagents for several weeks. These treatments oftenassisted with bleaching trays (night-guard) in order to better localizebleaching products and, thereby, maximize the whitening effect. However,even though these at home products contain active whitening agents inlower concentrations, similar side effects as those associated withprofessional treatments are very common. In addition, there arepaint-on, at-home whitening products, also known as “tooth varnishes”,as well as whitening strips intended to eliminate the need for dentaltrays. However, these products require more frequent applications,usually 3-times in a day, to complete whitening procedure. Finally,among the variety of at-home use products, whitening toothpastes andgels are the least effective form of whitening products due to theirshort contact time with the tooth surfaces. Although bleaching agentadditives augment the effectiveness, the whitening effect is primarilyas a result of removal of surface stains via mechanical action ofbrushing and other polishing ingredients (for example, silicaparticles).

Dental caries is one of the major public health problems and it is ahighly prevalent disease among the global population. Incipient cariesand white spot lesions (WSL) as well as hypersensitivity, are theearliest clinical evidence of enamel demineralization and dental caries.Dental caries occurs when tooth enamel is exposed to acid produced bycariogenic bacteria. As a result, acid diffuses into surface enamel anddissolves hydroxyapatite (HAp) mineral. Due to its non-regenerativenature, enamel is unable to heal and repair itselfpost-demineralization. Traditionally, fluoride (F) has been used as thekey agent in prevention of caries. Fluoride functions primarily viatopical mechanisms. It is believed that fluoride forms a thin layer ofnew but harder mineral, namely fluorapatite (FAp) which is incorporatedinto the existing HAp mineral on the tooth surface. There is a trend ofenhancing the remineralization effect of fluoride with calcium andphosphate supplementation in high risk individuals. Althoughcontroversial, the use of fluoride products remains the primarytreatment modality for caries prevention and remineralization, withmajor limitations regarding the efficacy of these products for thereversal or prevention of dental caries. Fluoride delivery systems,therefore, are not sufficient to overcome the high caries riskespecially in younger and elderly population.

There is presently an unmet need for tooth whitening and mineralizationmethods and products that reduce or eliminate the need for concentrated,abrasive oxidizing agents and attendant side effects such asdemineralization-associated tooth sensitivity and gum line injuries.

SUMMARY OF THE INVENTION

Herein we provide methods and compositions for whitening teeth where thenatural color of teeth is restored and improved upon by generating newlyformed thin mineral layer on discolored tooth surface using one or morebiomineralizing polypeptides. Herein we also provide methods andcompositions for mineralizing teeth.

In a first aspect, the present application provides a method forwhitening teeth, comprising administering to a subject in need thereofan amount effective to whiten the teeth of a biomineralizingpolypeptide. In some embodiments, the method for whitening teethcomprises administering to a subject in need thereof an effective amountof a composition comprising a biomineralizing polypeptide. In someembodiments, the biomineralizing polypeptide comprises an amino acidsequence selected from the group consisting of:

(ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP5; SEQ ID NO: 13)(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17)(LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG (H/Q)HSMTP(T/I)QH)₁₋₁₀;(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀; and 12-42 contiguous amino acids of(ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀;or a functional equivalent thereof, or any combination thereof. In someembodiments, the biomineralizing polypeptide comprises the amino acidsequence (PGYIN(L/F)SYE(K/N) SHSQAIN(T/V)DRTA)₁₋₁₀ (ADPS; SEQ ID NO:13),or a functional equivalent thereof. In some embodiments, thebiomineralizing polypeptide comprises the amino acid sequence(PGYINFSYENSHSQAINVDRTA)₁₋₁₀ (ADPSH; SEQ ID NO:15), or a functionalequivalent thereof. In some embodiments, the biomineralizing polypeptidecomprises the amino acid sequence (SYENSHSQAINVDRT)₁₋₁₀ (shADP5; SEQ IDNO:16), or a functional equivalent thereof. In some embodiments, thebiomineralizing polypeptide comprises(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQ QPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀(ADP7; SEQ ID NO:18), 12-42 contiguous amino acids of(HPP(S/T)HTLQPHHH(L/I) PVVPAQQPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀(ADP7), or a functional equivalent thereof. In some embodiments, thebiomineralizing polypeptide comprises an amino acid sequence selectedfrom the group consisting of:

(ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4)(VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀ (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG (H/Q)HSMTP(T/I)QH)₁₋₁₀;(ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3)(HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀;(ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11)(HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀;(ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀;(ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀;(ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23)(PAQQPVIPQQPMMP)₁₋₁₀;or a functional equivalent thereof, or any combination thereof. In someembodiments, the biomineralizing polypeptide comprises an amino acidsequence selected from the group consisting of:

(ADP3M; SEQ ID NO: 8) (WPATDKTKREEVD)₁₋₁₀; and (ADP3H; SEQ ID NO: 9)(WPSTDKTKREEVD)₁₋₁₀,or a functional equivalent thereof, or a combination thereof. In someembodiments, the biomineralizing polypeptide comprises one or morefusion peptides, wherein each of the one or more fusion peptidesindependently comprises two or more amino acid sequences selected fromthe group consisting of:

(ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4)(VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP3; SEQ ID NO: 7)(WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP5; SEQ ID NO: 13)(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17)(LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(ADP1M; SEQ ID NO: 2) (HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3)(HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀;(ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11)(HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀;(ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀;(ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀;(ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23)(PAQQPVIPQQPMMP)₁₋₁₀;or a functional equivalent thereof. In some embodiments, thebiomineralizing polypeptide further comprises a fluorescent agent. Insome embodiments, the method further comprises applying light to theteeth of the subject, thereby further whitening the teeth. In someembodiments, the light is selected from one of a diode laser; a PAClight; and a halogen light. In some embodiments, the method furthercomprises administering at least one cleaning agent to the teeth of thesubject. In some embodiments, the method further comprises administeringhydrogen peroxide, carbamide peroxide, titanium dioxide,nano-hydroxyapatite particles, zirconia powder, or any combinationthereof to the teeth of the subject.

In a second aspect, the present application provides an oral careproduct, comprising at least one biomineralizing polypeptide, at leastone calcium ion source, and at least one phosphate ion source. The oralcare product can further comprise at least one cleaning agent. In someembodiments, the at least one cleaning agent is selected from the groupconsisting of hydrogen peroxide, titanium dioxide, carbamide peroxide,nano-hydroxyapatite particles, zirconia powder, or any combinationthereof. The calcium ion source is generally any calcium salt. In someembodiments, the calcium ion source is selected from the groupconsisting of calcium acetate, calcium carbonate, calcium citrate,calcium chloride, calcium gluconate, calcium glycerophosphate, calciumlactate, and calcium phosphate. The phosphate ion source is generallyany phosphate salt. In some embodiments, the phosphate ion source isselected from the group consisting of aluminum phosphates, calciumphosphates, potassium phosphates, and sodium phosphates. In someembodiments, the oral care product is selected from the group consistingof toothpaste, toothpowders, mouthwash, gel, dental floss, liquiddentifrices, dental tablets, topical gels, troches, chewing gums, dentalpastes, gingival massage creams, gargle tablets, lozenges, tooth trays,tooth varnishes, and food products. In some embodiments, thebiomineralizing polypeptide comprises an amino acid sequence selectedfrom the group consisting of:

(ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP5; SEQ ID NO: 13)(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17)(LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; and (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀; and 12-42 contiguous amino acids of(ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀;

or a functional equivalent thereof, or any combination thereof. In someembodiments, the biomineralizing polypeptide comprises the amino acidsequence (PGYINFSYENSHSQAINVDRTA)₁₋₁₀ (ADPSH; SEQ ID NO:15), or afunctional equivalent thereof. In some embodiments, the biomineralizingpolypeptide comprises the amino acid sequence (SYENSHSQAINVDRT)₁₋₁₀(shADP5; SEQ ID NO:16), or a functional equivalent thereof. In someembodiments, the biomineralizing polypeptide comprises(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQ QPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀(ADP7; SEQ ID NO:18), 12-42 contiguous amino acids of(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I) PQQPMMPVPG(H/Q)HSMTP (T/I)QH)₁₋₁₀(ADP7; SEQ ID NO:18), or a functional equivalent thereof. In someembodiments, the biomineralizing polypeptide comprises an amino acidsequence selected from the group consisting of:

(ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4)(VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3)(HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀;(ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11)(HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀;(ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀;(ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀;(ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23)(PAQQPVIPQQPMMP)₁₋₁₀;

or a functional equivalent thereof, or any combination thereof.

In another aspect, the present application provides methods formineralizing teeth, comprising administering to one or more teeth of asubject in need thereof an effective amount to mineralize teeth of apolypeptide comprising or consisting of the amino acid sequence selectedfrom the group consisting of:

(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀; (ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀;(ADP5; SEQ ID NO: 13) (PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀;(ADP6; SEQ ID NO: 17) (LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀;(ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; and12-42 contiguous amino acids of (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQ QPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀;

or a functional equivalent thereof, or any combination thereof.

In various embodiments, the polypeptide comprises or consists of anamino acid sequence selected from the group consisting of:

(ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4)(VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3)(HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀;(ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11)(HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀;(ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀;(ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀;(ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23)(PAQQPVIPQQPMMP)₁₋₁₀; (ADP3M; SEQ ID NO: 8) (WPATDKTKREEVD)₁₋₁₀; and(ADP3H; SEQ ID NO: 9) (WPSTDKTKREEVD)₁₋₁₀,

or a functional equivalent thereof, or a combination thereof.

In various embodiment, the polypeptide comprises a fusion polypeptidecomprising two or more of the amino acid sequences selected from thegroup consisting of:

(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀ (ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀;(ADP2; SEQ ID NO: 4) (VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP3; SEQ ID NO: 7)(WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP5; SEQ ID NO: 13)(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17)(LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3)(HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀;(ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11)(HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀;(ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀;(ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀;(ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23)(PAQQPVIPQQPMMP)₁₋₁₀;

or a functional equivalent thereof.

In other embodiments, the polypeptide may be used as a single copy ofthe polypeptide.

In a further embodiment, the method further comprises administering tothe subject at least one calcium ion source and at least one phosphateion source. In various embodiments, the calcium ion source may beselected from the group consisting of calcium acetate, calciumcarbonate, calcium citrate, calcium chloride, calcium gluconate, calciumglycerophosphate, calcium lactate, and calcium phosphate. In variousother embodiments, the phosphate ion source may be selected from thegroup consisting of aluminum phosphates, calcium phosphates, potassiumphosphates, and sodium phosphates. In one embodiment, the methodcomprising administering a formulation of the polypeptide, at least onecalcium ion source and at least one phosphate ion source, wherein theformulation has

(a) a Ca²⁺ concentration ranging between about 1 mM and about 2 M, orbetween about 1 mM and about 1M, between about 1 mM and about 0.5M,between about 1 mM and about 100 mM, between about 1 mM and about 50 mM,between about 1 mM and about 10 mM, between about 2 mM and about 8 mM,between about 3 mM and about 7 mM, between about 4 mM and about 6 mM, orbetween about 4.5 mM and about 5.5 mM; and

(b) a PO₄ ³⁻ concentration ranging between about 0.5 mM and about 2 M,or between about 0.5 mM and about 1M, between about 0.5 mM and about0.5M, between about 0.5 mM and about 100 mM, between about 0.5 mM andabout 50 mM, between about 0.5 mM and about 10 mM, between about 0.5 mMand about 7 mM, or between about 1 mM and about 6 mM, between about 1.5mM and about 5 mM, between about 2 mM and about 4 mM, or between about2.5 mM and about 3.5 mM.

The methods may further comprise administering fluoride to the subject.In various embodiments, the fluoride may be present in the formulationat between about between about 50 parts per million (ppm) and about20,000 ppm, between about 50 ppm and about 10,000 ppm, between about 50ppm and about 5000 ppm, between about 50 ppm and about 1000 ppm, betweenabout 50 ppm and about 500 ppm, or about 75 ppm and about 400 ppm,between about 100 ppm and about 300 ppm, between about 150 ppm and about250 ppm, or about 200 ppm.

In other embodiments, the polypeptides and any ion sources and/orfluoride are administered in a formulation, and the formulation may be,but is not limited to, toothpaste, toothpowders, mouthwash, gel, dentalfloss, liquid dentifrices, dental tablets, topical gels, troches,chewing gums, dental pastes, gingival massage creams, gargle tablets,lozenges, tooth trays, tooth varnishes, and food products. In specificembodiments, the formulation may comprise a lozenge, a gel, ormouthwash.

In other embodiments, the polypeptide is administered in a formulationand is present in the formulation at a concentration of between about0.01 mM and about 0.5M, or between about 0.01 mM and about 0.1M, betweenabout 0.01 mM and about 50 mM, between about 0.01 mM to about 20 mM, orbetween about 0.1 mM to about 15 mM, or between about 0.5 mM to about12.5 mM, or between about 0.8 mM and about 10 mM.

In one embodiment, the subject is one suffering from demineralization ofenamel, typically characterized as incipient carious lesions, of one ormore tooth. In other embodiments, the subject has an incipient cariouslesion, tooth hypersensitivity, and/or white spot lesion (a clinicalcondition that is a result of demineralization of enamel), and theeffective amount is an amount that is effective to treat the incipientcarious lesions, tooth hypersensitivity, and/or white spot lesion.

In a further aspect, the invention provides polypeptide consisting ofthe amino acid sequence of

(shADP5; SEQ ID NO:16) (SYENSHSQAINVDRT)₁₋₁₀; or (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀.

In another aspect, the invention provides oral care products, comprising

(a) (SYENSHSQAINVDRT)₁₋₁₀ (shADP5; SEQ ID NO:16); and/or(SYEKSHSQAINTDRT)₁₋₁₀ (sADP5; SEQ ID NO:24);

(b) at least one calcium ion source, and

(c) at least one phosphate ion source.

In various embodiments, the calcium ion source may be selected from thegroup consisting of calcium acetate, calcium carbonate, calcium citrate,calcium chloride, calcium gluconate, calcium glycerophosphate, calciumlactate, and calcium phosphate. In various further embodiments, thephosphate ion source is selected from the group consisting of aluminumphosphates, calcium phosphates, potassium phosphates, and sodiumphosphates. In various further embodiments, the oral care product has

(i) a Ca²⁺ concentration ranging between about 1 mM and about 2 M, orbetween about 1 mM and about 1M, between about 1 mM and about 0.5M,between about 1 mM and about 100 mM, between about 1 mM and about 50 mM,between about 1 mM and about 10 mM, between about 2 mM and about 8 mM,between about 3 mM and about 7 mM, between about 4 mM and about 6 mM, orbetween about 4.5 mM and about 5.5 mM; and

(ii) a PO₄ ³⁻ concentration ranging between about 0.5 mM and about 2 M,or between about 0.5 mM and about 1M, between about 0.5 mM and about0.5M, between about 0.5 mM and about 100 mM, between about 0.5 mM andabout 50 mM, between about 0.5 mM and about 10 mM, between about 0.5 mMand about 7 mM, or between about 1 mM and about 6 mM, between about 1.5mM and about 5 mM, between about 2 mM and about 4 mM, or between about2.5 mM and about 3.5 mM.

In a further embodiment, the oral care product may further comprisefluoride. In various non-limiting embodiments, the fluoride may bepresent in the oral care product at between about between about 50 partsper million (ppm) and about 20,000 ppm, between about 50 ppm and about10,000 ppm, between about 50 ppm and about 5000 ppm, between about 50ppm and about 1000 ppm, between about 50 ppm and about 500 ppm, or about75 ppm and about 400 ppm, between about 100 ppm and about 300 ppm,between about 150 ppm and about 250 ppm, or about 200 ppm.

In various embodiments, the oral care product may be selected from thegroup consisting of toothpaste, toothpowders, mouthwash, gel, dentalfloss, liquid dentifrices, dental tablets, topical gels, troches,chewing gums, dental pastes, gingival massage creams, gargle tablets,lozenges, tooth trays, tooth varnishes, and food products. In specificembodiments, the oral care product comprises a lozenge a gel, ormouthwash.

In other embodiments, the polypeptide may be present in thepharmaceutical composition at a concentration of between about 0.01 mMand about 0.5M, or between about 0.01 mM and about 0.1M, between about0.01 mM and about 50 mM, between about 0.01 mM to about 20 mM, orbetween about 0.1 mM to about 15 mM, or between about 0.5 mM to about12.5 mM, or between about 0.8 mM and about 10 mM.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thedescribed technology will become more readily appreciated as the samebecome better understood by reference to the following detaileddescription, when taken in conjunction with the accompanying drawings,wherein:

FIG. 1 depicts a tooth (a) before and (b) after sectioning vertically asdescribed in Example 2.

FIG. 2 depicts various sections of a tooth after staining as describedin Example 2. (a) shows one half of a sectioned tooth after thestaining. (b), (c) and (d) show different views of the left quarter ofthe tooth that was sectioned from the half tooth. (e), (f) and (g) showdifferent views of the right quarter of the tooth that was sectionedfrom the half tooth.

FIG. 3 shows the left quarter (control) and the right quarter of thetooth (test tooth) as described in Example 2.

FIG. 4 depicts a line profile analysis of a portion of the originaltooth (before staining), a portion of the tooth after staining, and aportion of tooth after staining and remineralization, as described inExample 2.

FIG. 5 depicts a tooth before staining, a side-by-side comparison of astained tooth portion with a 3-rounds remineralized tooth portion, aside-by-side comparison of a stained tooth portion with a 4-roundsremineralized tooth portion, a side-by-side comparison of a stainedtooth portion with a 5-rounds remineralized tooth portion, and aside-by-side comparison of a stained tooth portion with a 7-roundsremineralized tooth portion, as described in Example 8.

FIG. 6 shows a line profile analysis of the tooth before staining (BarA), the 3-rounds remineralized tooth (Bar B), the 4-rounds remineralizedtooth (Bar C), the 5-rounds remineralized tooth (Bar D), and the7-rounds remineralized tooth (Bar E), as described in Example 8.

FIG. 7. (a) White spot lesion was artificially created by exposing awindow on tooth surface for demineralization; (b) Groups 5 & 6 sampleswere exposed to shADP5 solution for 10 minutes at 37° C.; (c) Sampleswere then incubated in F/Ca²⁺/PO₄ ³⁻ or Ca²⁺/PO₄ ³⁻ solutions for 1 hourat 37° C.; (d) New mineral layer was characterized by SEM/EDXS.

FIG. 8. (a) and (b)Face-on and (d) edge-on (c) SEM images and EDXSanalyses of Group 1: Negative Control. (e) and (f) Face-on and (h)edge-on SE images and EDXS analysis of Group 2: Ca′ and PO₄ ³⁻ only.Insets in 2b and 2f show enamel rods and HAp plate-like crystallitesexposed on the surface of damaged enamel as a result ofdemineralization. The inset panels are 1 μm×1 μm.

FIG. 9. (a) and (b) Face-on and (d)edge-on (c)SEM images and EDXSanalysis of Group 3: 1100 ppm F+Ca2+/PO₄3⁻. (e) and (f) Face-on and (h)edge-on SEM images and EDXS analysis

of Group 4: 20,000 ppm F+Ca2+/PO₄3⁻. Insets in 3b and 3f show looselypacked nanospherical particles (of dia. ˜20-30 nm) as a result of Fdeposition. The inset panels are 1 μm×1 μm.

FIG. 10. (a) and (b) Face-on and (d) edge-on (c) SEM images and EDXSanalyses of Group 5: shADP5+1100 ppm F+Ca²⁺/PO₄ ³⁻. Insets in (b) showloosely crystallized regions of accumulated 100-nm dia. sphericalnanoparticles on the surface. (e) and (f) Face-on and (h) edge-on SEMimages and EDXS analysis (g) of Group 6: shADP5+Ca²⁺/PO₄ ³⁻. Inset (f)displays a highly uniform, plate-like HAp crystallites within (h) newlyformed mineral layer in shADP5+Ca²⁺/PO₄ ³⁻ treatment. The inset panelsare 1 μm×1 μm.

FIG. 11. TEM bright field images and corresponding selected diffractionfor (a), (b) and (c) no treatment negative control (group 1), (d), (e)and (f) high concentration F treatment (group 4), and (g), (h) and (i)peptide treatment (group 6).

FIG. 12. (a) Atomic force microscope images of the surfaces of themineralized layers in samples from Group 2 (left) where there is noapparent mineral layer on the lesion and Group 6 (right) showing a clearboundary between the lesion and newly formed mineral layer (arrows). (b)Hardness (left) and elastic modulus (right) of the experimental groupsused here as measured by nanoindentation, n>20.

FIG. 13. TEM bright field images (top—low magnification, and middle,high magnification) and corresponding selected diffraction patterns(bottom row) showing the indices of the diffractions peaks. All top rowimages were recorded at the same magnifications, and middle ones werealso at the same high magnification, therefore micron size marks are thesame for top row and also for the middle row. The panels correspond to:(a), (b) and (c) No-treatment negative control, Group 1 (d), (e) and (f)Group 2 Ca and PO₄ ions only; (g), (h) and (i) Group 3, low Fluorideconcentration (1,100 ppm); (j), (k) and (l) Group 4, High Fconcentration (20,000 ppm)), (m), (n) and (o) Group 5, shADP5+low Fconcentration (1,100 ppm), and (p), (q) and (r) Group 6, shADP5 only.

FIG. 14. Schematics of (a) microhardness and (b) nanoindentation tests.

FIG. 15. Stained teeth before & after whitening treatments usingcommercial versus biomineralization methods. (a), (b) and (c) Theuntreated, tea-stained group (baseline). HP-based whitening products, byCREST® 3D Whitening™ strips (d) and (e) containing 14% HP, and byOPALESCENCE® Whitening gel containing 30% HP (f) and (g). The colorshade was increased compared to baseline by 5.7% and 6.8%, respectively.Compared to the tea stained teeth, not only was whitening is increasedwith peptide-treatment (h) and (i) by 4.6%, but also with the benefit ofremineralization. The SEM images in the insets display the details ofthe tooth surface showing the effects of etching effect of thecommercial products resulting in the rough surface in (e) & (g) whilenew mineralized layer formed by the peptide treatment in (i).

FIG. 16. Representative SEM images of hypersensitive teeth surfacebefore (first and second rows) and after (third and fourth rows) peptideguided remineralization treatment.

DETAILED DESCRIPTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R.I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term,or may mean plus or minus 10% of the particular term.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) providedherein, is intended merely to better illuminate the embodiments and doesnot pose a limitation on the scope of the claims unless otherwisestated. No language in the specification should be construed asindicating any non-claimed element as essential.

All embodiments of any aspect of the described technologies can be usedin combination, unless the context clearly dictates otherwise.

In a first aspect, methods for whitening teeth or for mineralizingteeth, are provided, comprising administering to a subject in needthereof an amount effective to whiten teeth or to mineralize teeth of abiomineralizing polypeptide or pharmaceutical composition or oral careproduct of any embodiment or combination of embodiments of thetechnologies. The methods can further comprise administering at leastone calcium ion source and at least one phosphate ion source to thesubject. The calcium ion source and phosphate ion source can beco-administered or serially administered. The calcium ion source andphosphate ion source can be administered before the biomineralizingpolypeptide, concurrently with the biomineralizing polypeptide, or afterthe biomineralizing polypeptide.

As shown herein, the inventors have discovered that biomineralizingpolypeptides can be used to whiten teeth and to remineralize toothenamel. While not being bound by a specific mechanism of action, theinventors believe that the polypeptides direct mineralization of dentallesions to form a “dento-mimetic” mineral layer, which also serves toreduce dental hypersensitivity and bacterial infiltration. It is furtherbelieved that some of the polypeptides exert their activity via bindingto hydroxyapatite (HA) surfaces on the tooth to kinetically promotere-mineralization, occluding of dentin tubules to prevent/limitstimulants from reaching the tubules, and/or rebuilding lost mineral tocreate a physical barrier against bacteria. The inventors furtherdemonstrate that crystalline mineral layer can be formed on enamellesions in the presence of Ca²⁺ and PO₄ ³⁻ ions together withpolypeptides of the invention under physiologically viable conditions.The inventors also demonstrate that the methods can promotelayer-by-layer mineralization, i.e., more layers as the treatment isapplied showing that the number of layers increases. The inventorsfurther demonstrate that use of the polypeptides of the invention allowthe delivery and incorporation of fluoride ions into the remineralizedlayer even at F concentrations much lower than those used in currentdental practice and products. Thus, the invention provides a greatimprovement over previously reported methods for treating dentaldisease.

The methods of the present application have a number of attendantadvantages. The described methods clear stains present on tooth surfacesin addition to re-mineralizing tooth surfaces. Without clearing stainsany re-mineralization would create incipient lesions on the tooth. Thedescribed methods lead to whitened and smooth tooth surfaces in contrastto conventional whiteners which may lead to uneven tooth surfaces, with“hills” and “valleys”.

As used herein, a “biomineralizing polypeptide” is any polypeptidecapable of generating a hydroxyapatite layer on a tooth in the presenceof calcium ions and phosphate ions. Several biomineralizing polypeptidesare described in PCT application No. PCT/US2012/039650, which isincorporated herein by reference in its entirety. The calcium ions arepresent due to at least one calcium ion source. The calcium ion sourcecan generally be any calcium salt. Illustrative calcium ion sourcesinclude, but are not limited to, calcium acetate, calcium carbonate,calcium citrate, calcium chloride, calcium gluconate, calciumglycerophosphate, calcium lactate, and calcium phosphate. In some cases,combinations of more than one calcium ion source may be used. In someembodiments, the calcium ion source is not calcium phosphate. Thephosphate ions are present due to at least one phosphate ion source. Thephosphate ion source can generally be any phosphate salt. Illustrativephosphate ion sources include, but are not limited to, aluminumphosphates, calcium phosphates, potassium phosphates, and sodiumphosphates. Calcium phosphates include monocalcium phosphate, dicalciumphosphate, and tricalcium phosphate. Potassium phosphates includemonopotassium phosphate, dipotassium phosphate, and tripotassiumphosphate. Sodium phosphates include sodium dihydrogen phosphate, sodiumhydrogen phosphate, and trisodium phosphate. In some cases, combinationsof more than one phosphate ion source may be used. In some embodiments,the phosphate ion source is not calcium phosphate. The concentration ofcalcium ions can generally be any concentration, such as about 0.1 mM toabout 100 mM. In various further embodiments, the calcium ions are usedat a concentration of between about 1 mM and about 2 M, or between about1 mM and about 1M, between about 1 mM and about 0.5M, between about 1 mMand about 100 mM, between about 1 mM and about 50 mM, between about 1 mMand about 10 mM, between about 2 mM and about 8 mM, between about 3 mMand about 7 mM, between about 4 mM and about 6 mM, or between about 4.5mM and about 5.5 mM. In various further embodiments, the phosphate ionsare used at a concentration ranging between about 0.5 mM and about 2 M,or between about 0.5 mM and about 1M, between about 0.5 mM and about0.5M, between about 0.5 mM and about 100 mM, between about 0.5 mM andabout 50 mM, between about 0.5 mM and about 10 mM, between about 0.5 mMand about 7 mM, or between about 1 mM and about 6 mM, between about 1.5mM and about 5 mM, between about 2 mM and about 4 mM, or between about2.5 mM and about 3.5 mM. The ratio of calcium ions to phosphate ions cangenerally be any ratio, such as about 5:3. In various embodiments,higher ionic concentrations may be used when gel formulations areemployed (in non-limiting embodiments, 0.1 M or more), while lower ionicconcentrations may be used when solution formulations are employed (innon-limiting embodiments, 50 mM or less).

In some embodiments, biomineralizing polypeptides comprise or consist ofone or more amelogenin derived polypeptides (ADPs).

In one embodiment, biomineralizing polypeptides comprise or consist of

(ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP5; SEQ ID NO: 13)(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17)(LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP7; SEQ ID NO: 18)(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀;(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀; and 12-42 contiguous amino acids of(ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀;or functional equivalents thereof. In various embodiments, the recitedpolypeptides may be present in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies,or ranges such as 2-10. For example, a tetrapeptide can be used thatcontains four consecutive copies of ADP6. In some embodiments, thebiomineralizing polypeptides comprise or consist of two or more of theADPs or functional equivalents thereof. In some embodiments, thebiomineralizing polypeptides comprise one or more fusion peptidescomprising or consisting of two or more of the ADPs or functionalequivalents thereof.

As used herein, a “functional equivalent” of a polypeptide is one thatretains the biological activity of the polypeptide in treating dentaldisease, and includes one or more amino acid substitutions, deletions,additions, or insertions. In various embodiments, the functionalequivalent is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, or more identical to the recited polypeptide. Insome embodiments, the functional equivalent is a shortened version of anADP described herein.

In another aspect, the invention provides a polypeptide consisting ofthe amino acid sequence of

(shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; or (sADP5; SEQ ID NO: 24)(SYEKSHSQAINTDRT)₁₋₁₀.

The inventors have discovered that these polypeptides of the inventionhave significantly enhanced aqueous solubility compared to other ADPSpolypeptides described to date, while retaining the mineralizationdirecting properties of ADPS. Thus, the polypeptides of this aspect ofthe invention provide a significant improvement over previousbiomineralization peptides. The polypeptide may be present in 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 copies; in one specific embodiment, thepolypeptide is present in one copy. While the polypeptides consist ofthe recited amino acid sequence, the invention further provides fusionproteins comprising a polypeptide of this aspect of the invention fusedto another polypeptide, formulations comprising the polypeptides of thisaspect of the invention and one or more calcium and/or phosphate ionsources, and various oral care products comprising the polypeptides ofthis aspect of the invention, as described herein.

As used throughout the present application, the term “polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids, whether naturally occurring or of synthetic origin. Thepolypeptides may comprise L-amino acids, D-amino acids (which areresistant to L-amino acid-specific proteases in vivo), or a combinationof D- and L-amino acids. The polypeptides described herein may bechemically synthesized or recombinantly expressed. The polypeptides maybe linked to other compounds to promote an increased half-life in vivo,such as by PEGylation, HESylation, PASylation, or glycosylation. Suchlinkage can be covalent or non-covalent as is understood by those ofskill in the art.

In one embodiment, the methods comprise administering a polypeptidecomprising or consisting of the amino acid sequence(PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀ (ADPS; SEQ ID NO:13), or afunctional equivalent thereof. In an alternative embodiment, ADPScomprises or consists of (PGYINFSYENSHSQAINVDRTA)₁₋₁₀ (ADPSH; SEQ IDNO:15), or a functional equivalent thereof. In another alternativeembodiment, ADPS comprises or consists of (PGYINLSYEKSHSQAINTDRTA)₁₋₁₀(ADPSM; SEQ ID NO:14), or a functional equivalent thereof. In yetanother alternative embodiment, ADPS comprises or consists of(SYENSHSQAINVDRT)₁₋₁₀ (shADP5; SEQ ID NO:16), or a functional equivalentthereof. In an alternative embodiment, ADPS comprises or consists of(SYEKSHSQAINTDRT)₁₋₁₀ (sADP5; SEQ ID NO:24) or a functional equivalentthereof. In various embodiments, ADPS, ADPSM, shADP5, sADP5, or ADPSH ispresent in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies, or ranges such as2-10. In an alternative embodiment, ADPS, ADPSM, shADP5, sADP5, or ADPSHis present in 1 copy. In yet another alternative embodiment, the ADPScomprises or consists of ADPSH or shADP5, preferably in one copy. In yetanother alternative embodiment, the ADPS comprises or consists of sADP5,preferably in one copy. In some embodiments, the biomineralizingpolypeptides comprise or consist of two or more of the ADPs orfunctional equivalents thereof. In some embodiments, the biomineralizingpolypeptides comprise one or more fusion peptides comprising orconsisting of two or more of the ADPs or functional equivalents thereof.

In another embodiment, the methods comprise administering a polypeptidecomprising or consisting of the amino acid sequence:(HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV (A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀(ADP7; SEQ ID NO:18), or a functional equivalent thereof. In anembodiment, the ADP7 comprises or consists of(HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀ (ADP7M; SEQ ID NO:19);(HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀ (ADP7H; SEQ ID NO:20),or a functional equivalent thereof. In various embodiments, ADP 7,ADP7M, or ADP7H is present in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies,or ranges such as 2-10. In an embodiment, ADP7, ADP7M, or ADPH7 ispresent in 1 copy. In another embodiment, the ADP7 comprises or consistsof ADP7H in one copy. In some embodiments, the biomineralizingpolypeptides comprise or consist of two or more of the ADPs orfunctional equivalents thereof. In some embodiments, the biomineralizingpolypeptides comprise one or more fusion peptides comprising orconsisting of two or more of the ADPs or functional equivalents thereof.

In a further embodiment, the polypeptide for use in the methodscomprises or consists of the amino acid sequence WP(A/S)TDKTKREEVD)₁₋₁₀(ADP3; SEQ ID NO:7), or a functional equivalent thereof. In anembodiment, ADP3 comprises or consists of ((WPATDKTKREEVD)₁₋₁₀ (ADP3M;SEQ ID NO:8) or (WPSTDKTKREEVD)₁₋₁₀ (ADP3H; SEQ ID NO:9), or afunctional equivalent thereof. In various embodiments, ADP3, ADP3M, orADP3H is present in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies, or rangessuch as 2-10. In an embodiment, ADP3, ADP3M, or ADP3H is present in 1copy. In another embodiment, the ADP3 comprises or consists of ADP3H inone copy. In some embodiments, the biomineralizing polypeptides compriseor consist of two or more of the ADPs or functional equivalents thereof.In some embodiments, the biomineralizing polypeptides comprise one ormore fusion peptides comprising or consisting of two or more of the ADPsor functional equivalents thereof.

In various further embodiments, the polypeptide for use in the methodscomprises or consists of a polypeptide selected from the groupconsisting of selected from the group consisting of:

(ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4)(VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP4; SEQ ID NO: 10)(HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP8; SEQ ID NO: 21)(PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP1M; SEQ ID NO: 2) (HTLQPHHHLPVV)₁₋₁₀;(ADP1H; SEQ ID NO: 3) (HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5)(VPGHHSMTPTQH)₁₋₁₀; (ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀;(ADP4M; SEQ ID NO: 11) (HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12)(HPPTHTLQPHHHIPVV)₁₋₁₀; (ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and(ADP8H; SEQ ID NO: 23) (PAQQPVIPQQPMMP)₁₋₁₀;

or functional equivalents thereof.

In various embodiments, the recited polypeptides may be present in 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 copies, or ranges such as 2-10. In someembodiments, when present in more than one copy, the copies arecontiguous to each other. In some embodiments, the polypeptide for usein the methods comprises a fusion of two or more of the ADPs describedherein or functional equivalents thereof.

Each ADP and functional equivalent thereof has its own distinct kineticprofile and may exhibit a fast or slow kinetic profile. For example,ADPS has a fast kinetic profile whereas ADP7 has a slow kinetic profile.In some embodiments, the biomineralizing polypeptide comprises orconsists of at least one ADP with a slow kinetic profile and at leastone ADP with a fast kinetic profile. In some embodiments, thebiomineralizing polypeptide only comprises or consists of one or moreADPs with fast kinetic profiles. In some embodiments, thebiomineralizing polypeptide only comprises or consists of one or moreADPs with slow kinetic profiles. Fast kinetics refers to fast mineralformation, such as immediate mineral formation, while slow mineralformation refers to mineral formation over a period of time, such asabout 5 minutes to about 1 hour. In some embodiments, a firstbiomineralizing peptide having faster kinetics can be used incombination with a second biomineralizing peptide having slowerkinetics.

In some embodiments, the polypeptides of the invention or thepolypeptide for use in the methods further comprises at least onefluorescent agent. In some embodiments, the polypeptides of theinvention or the polypeptide for use in the methods comprises apolypeptide covalently linked to at least one fluorescent agent.Illustrative fluorescent agents include, but are not limited to,fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594,tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6F,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight®350, DyLight® 405, DyLight® 488, DyLight® 549, DyLight® 594, DyLight®633, DyLight® 649, DyLight® 680, DyLight® 750, DyLight® 800,phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6-)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, OregonGreen 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, AlexFluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, AlexaFluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid,BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530/550, BODIPY® 558/568, BODIPY®630/650, BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, andcombinations thereof.

The subject may be any subject whose teeth could be whitened orremineralized, including but not limited to mammals. In variousembodiments, the mammal is a human, dog, cat, horse, cow, sheep, goat,pig, or other pet or food/dairy animal. In one embodiment, the subjectis a human. In one embodiment, the subject is suffering fromdemineralization of enamel and/or dentin of one or more tooth. Forexample, the subject may have one or more of incipient carious lesion,tooth hypersensitivity, and/or white spot lesion, and the effectiveamount administered is an amount of the polypeptide and ions of Ca andPO₄ that is effective to treat the one or more incipient carious lesion,tooth hypersensitivity, and/or white spot lesion.

As used herein, “tooth whitening” and “dental bleaching” have identicalmeanings and can be used interchangeably. Tooth whitening and dentalbleaching refer to decreasing the amount of chromagens or discolorantspresent in or on a tooth. In certain embodiments, tooth whitening refersto restoring a tooth to its original color. In certain otherembodiments, dental bleaching refers to whitening a tooth beyond itsnatural, original color. Tooth whitening may be measured using methodsknown to those skilled in the art. In some embodiments, tooth whiteningis measured using comparative shade guides. In some embodiments, toothcolor and tooth whitening are measured using a colorimeter, such as atristimulus colorimeter. Such instruments can be used to measure thecolor of the specimen (tooth) surface quantitatively. The CIE “L*a*b*”color system is commonly used in the commercial and scientificliterature for quantitatively measuring teeth color (See Yiming, J.Esther Restor. Dent., 15: 533-541 (2003)). The system uses one luminanceparameter and two color coordinates to specify a point on a chromaticitydiagram. The “L” value corresponds to lightness, the “a” valuecorresponds to red-green, and the “b” value corresponds to yellow-blue.A change in the color of a treated tooth can be calculated asΔE_(Lab)=((ΔL*)² (Δa*)²+(Δb*)²)^(1/2). The value can generally be anyvalue, and in some cases have a minimum value limited only by thedetection limit of the instrument used to take the measurements.

As used herein, “mineralizing” or “remineralizing” are usedinterchangeably, and mean mineral layer formation on damaged enamel.Thus, subjects in need of mineralization are those that have damage tothe enamel that can benefit from the mineralization methods of theinvention. The methods may result in any amount of mineralization, asany such mineralization provides a therapeutic benefit to the subject.In various embodiments the mineralization may result in production of amineral layer at least 1 μm on the tooth enamel; in other embodiments,the methods result in production of a mineral layer at least 2, 3, 4, 5,6, 7, 8, 9, or 10 μm on the tooth enamel. In one embodiment, the minerallayer produced on the enamel comprises a dense mineralized layercontaining hydroxyapatite, also known as hydroxyl apatite (Hap). Inother embodiments, the newly formed mineral layer is integrated with theunderlying enamel.

As used herein, unless otherwise described, an “amount effective” or“effective amount” refers to an amount of the polypeptide that iseffective for whitening teeth and/or for mineralizing teeth. In someembodiments, the “amount effective” or “effective amount” means aconcentration of about 0.01 mM to about 1 mM of the polypeptide. Thisincludes a concentration of about 0.05 mM to about 0.5 mM or about 0.1mM to about 0.5 mM. In some embodiments, the concentration is about0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20,0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80,0.85, 0.90, 0.95, or 1 mM, including increments therein, of polypeptide.In a clinical setting, higher concentrations of polypeptide (forexample, about 0.6 mM to about 0.8 mM) may be used. In a home usesetting, lower concentrations of polypeptide (for example, about 0.01 mMto about 0.1 mM) may be used. In one embodiment, the polypeptide isadministered in a formulation and is present in the formulation at aconcentration of about 0.01 mM to about 20 mM; in various furtherembodiments, the polypeptide is present in the formulation at betweenabout 0.01 mM and about 0.5M, or between about 0.01 mM and about 0.1M,between about 0.01 mM and about 50 mM, between about 0.01 mM to about 20mM, or between about 0.1 mM to about 15 mM, or between about 0.5 mM toabout 12.5 mM, or between about 0.8 mM and about 10 mM. In someembodiments, the “amount effective” or “effective amount” refers to anamount of a composition comprising the polypeptide that is effective forwhitening teeth and/or for mineralizing teeth.

The methods may further comprise administering fluoride to the subject.In various embodiments, the fluoride may be present in the formulationat between about 50 ppm and about 20,000 ppm, or between about 50 ppmand about 10,000 ppm, between about 50 ppm and about 5000 ppm, betweenabout 50 ppm and about 1000 ppm, between about 50 ppm and about 500 ppm,or about 75 ppm and about 400 ppm, between about 100 ppm and about 300ppm, between about 150 ppm and about 250 ppm, or about 200 ppm. Theexamples demonstrate that use of the polypeptides of the invention allowthe delivery and incorporation of fluoride ions into the remineralizedlayer even at fluoride concentrations much lower than those used incurrent dental practice and products.

The polypeptides are typically formulated as a pharmaceuticalcomposition, such as those disclosed herein, and can be administered viaany suitable route, including orally, parentally, by inhalation spray,or topically in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles.

In an embodiment, the pharmaceutical compositions and formulations aretopically administration, such as in the form of ointments, lotions,creams, pastes, gels, drops, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be included.

In certain preferred embodiments, the polypeptides are delivered by:placing the polypeptides in contact with a tooth surface and applying alight source to the tooth. Without being bound by theory, it is believedthat a light source can increase the rate of tooth whitening and/or formineralizing. Such light sources can include diode lasers, PAC lights,and halogen lights.

In certain preferred embodiments, the polypeptides are delivered byplacing the polypeptides in contact with a tooth surface also in thepresence of at least one cleaning agent. Examples of cleaning agentsinclude hydrogen peroxide, carbamide peroxide, titanium dioxide,nano-hydroxyapatite particles, zirconia powder, and combinationsthereof.

In certain preferred embodiments, the biomineralizing polypeptides arepresent in a gel or other pharmaceutical composition, as describedherein, placed in a mouth guard and applied to the teeth of a subjectovernight. Such repeated overnight application of biomineralizingpolypeptides can have especially beneficial whitening and/or formineralizing effects.

Similarly, the biomineralizing polypeptides described herein can beapplied to the teeth of a subject as a mouthwash, toothpaste, or toothvarnish on a daily basis to similar effect.

In some embodiments, the pharmaceutical compositions used in the methodsdescribed herein comprise at least one biomineralizing polypeptide and acalcium ion source. In some embodiments, the pharmaceutical compositionsused in the methods described herein comprise at least onebiomineralizing polypeptide and a phosphate ion source. In someembodiments, the pharmaceutical compositions used in the methodsdescribed herein comprise at least one biomineralizing polypeptide, acalcium ion source, and a phosphate ion source.

Dosage regimens can be adjusted to provide the optimum desired response(for example, a therapeutic or prophylactic response). A suitable dosagerange may, for instance, be about 0.1 μg/kg to about 100 mg/kg bodyweight; alternatively, it may be about 0.5 μg/kg to about 50 mg/kg;about 1 μg/kg to about 25 mg/kg, or about 5 μg/kg to about 10 mg/kg bodyweight. The polypeptides can be delivered in a single bolus, or may beadministered more than once (for example, 2, 3, 4, 5, or more times) asdetermined by an attending physician.

In various other embodiments, the heterologous polypeptide providesadded functionality, for example, when the fusion polypeptides are usedto whiten and/or mineralize teeth. Exemplary such heterologouspolypeptides include, but are not limited to biomineralization-promotingpolypeptides (that is, any other polypeptides that are useful forcontrolling or promoting biomineralization). As will be understood bythose of skill in the art, the recited heterologous polypeptide maycomprise or consist of the full length protein, or functionalpolypeptides derived therefrom. Such heterologous polypeptides are knownto those of skill in the art. A recombinant fusion protein can comprisethe ADP polypeptide and at least one heterologous polypeptide.

In a further aspect, pharmaceutical compositions are provided,comprising one or more polypeptides, recombinant fusion proteins, orcompositions with a pharmaceutically acceptable carrier. Thepharmaceutical compositions can be used, for example, in the methodsdescribed herein. The pharmaceutical composition may comprise inaddition to the polypeptide (a) at least one lyoprotectant; (b) at leastone surfactant; (c) at least one bulking agent; (d) at least onetonicity adjusting agent; (e) at least one stabilizer; (f) at least onepreservative; and/or (g) at least one buffer.

In some embodiments, the buffer in the pharmaceutical composition is aTris buffer, a histidine buffer, a phosphate buffer, a citrate buffer,or an acetate buffer. The pharmaceutical composition may also include atleast one lyoprotectant, for example, sucrose, sorbitol, or trehalose.In certain embodiments, the pharmaceutical composition includes at leastone preservative, for example, benzalkonium chloride, benzethonium,chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben,propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol,phenylmercuric nitrate, thimerosal, benzoic acid, or various mixturesthereof. In other embodiments, the pharmaceutical composition includesat least one bulking agent, such as glycine. In yet other embodiments,the pharmaceutical composition includes at least one surfactant, forexample, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65,polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate,sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate,sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or anycombination thereof. The pharmaceutical composition may also include atleast one tonicity adjusting agent, for example, a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine, andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes at least one stabilizer, for example,a molecule which, when combined with a protein of interest substantiallyprevents or reduces chemical and/or physical instability of the proteinof interest in lyophilized or liquid form. Exemplary stabilizers includesucrose, sorbitol, glycine, inositol, sodium chloride, methionine,arginine, and arginine hydrochloride.

The polypeptides may be the sole active agent in the pharmaceuticalcomposition, or the composition may further comprise one or more otheractive agents suitable for an intended use, including but not limited toantimicrobial polypeptides (inhibiting bacterial infection), otherbiomineralization-promoting polypeptides (that is, any otherpolypeptides that are useful for controlling or promotingbiomineralization), inorganic material-binding polypeptides,three-dimensional scaffold-forming polypeptides, collagen, chitosan,amphiphilic peptides, protein-binding polypeptides, enamelin-derivedpolypeptides, tuftelin-derived peptides, statherin-derived polypeptides,dentin-derived polypeptides, bone sialoprotein-derived polypeptides,osteocalcin-derived polypeptides, osteopontin-derived polypeptides,proteins with caries inhibitory activity, casein, and bonemorphogenetic-derived polypeptides.

The pharmaceutical compositions described herein generally comprise acombination of a compound described herein and a pharmaceuticallyacceptable carrier, diluent, or excipient. Such compositions aresubstantially free of non-pharmaceutically acceptable components, thatis, contain amounts of non-pharmaceutically acceptable components lowerthan permitted by regulatory requirements. In some embodiments of thisaspect, if the compound is dissolved or suspended in water, thecomposition further optionally comprises an additional pharmaceuticallyacceptable carrier, diluent, or excipient. In other embodiments, thepharmaceutical compositions described herein are solid pharmaceuticalcompositions (for example, tablet, capsules, lozenges, and so on).

These compositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by any suitable route. In anembodiment, the pharmaceutical compositions and formulations aredesigned for topical administration, and may include ointments, lotions,creams, pastes, gels, drops, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

The pharmaceutical compositions can be in any suitable form, includingbut not limited to tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to about10% by weight of the active compound, soft and hard gelatin capsules,sterile injectable solutions, and sterile packaged powders.

In certain embodiments, the pharmaceutical compositions comprise atleast one cleaning agent in addition to the biomineralizingpolypeptides. These cleaning agents can include hydrogen peroxide,carbamide peroxide, titanium dioxide, nano-hydroxyapatite particles,zirconia powder, or combinations thereof. The pharmaceuticalcompositions can also include abrasive agents, such as silica particles.In some embodiments, the pharmaceutical compositions further comprise acalcium ion source, a phosphate ion source, or both.

In one embodiment the pharmaceutical compositions are in the form of anoral care product, including but not limited to toothpaste,toothpowders, mouthwash, dental floss, liquid dentifrices, dentaltablets, topical gels, troches, chewing gums, dental pastes, gingivalmassage creams, gargle tablets, lozenges, and food products. Thus, inanother aspect, oral care products are provided, comprising anyembodiment or combination of embodiments of the polypeptides,recombinant fusion proteins, and/or compositions. Such oral careproducts can be used, for example, in whitening and/or mineralizingteeth. In some embodiments, provided herein are oral care productscomprising at least one biomineralizing polypeptide, at least onecalcium ion source, at least one phosphate ion source, and at least onecleaning agent. The calcium ions are present due to at least one calciumion source. The calcium ion source can generally be any calcium salt.Illustrative calcium ion sources include, but are not limited to,calcium acetate, calcium carbonate, calcium citrate, calcium chloride,calcium gluconate, calcium glycerophosphate, calcium lactate, andcalcium phosphate. In some cases, combinations of more than one calciumion source may be used. In some embodiments, the calcium ion source isnot calcium phosphate. The phosphate ions are present due to at leastone phosphate ion source. The phosphate ion source can generally be anyphosphate salt. Illustrative phosphate ion sources include, but are notlimited to, aluminum phosphates, calcium phosphates, potassiumphosphates, and sodium phosphates. Calcium phosphates includemonocalcium phosphate, dicalcium phosphate, and tricalcium phosphate.Potassium phosphates include monopotassium phosphate, dipotassiumphosphate, and tripotassium phosphate. Sodium phosphates include sodiumdihydrogen phosphate, sodium hydrogen phosphate, and trisodiumphosphate. In some cases, combinations of more than one phosphate ionsource may be used. In some embodiments, the phosphate ion source is notcalcium phosphate. The concentration of calcium ions can generally beany concentration, such as about 0.1 mM to about 100 mM. In variousfurther embodiments, the calcium ions are used at a concentration ofbetween about 1 mM and about 2 M, or between about 1 mM and about 1M,between about 1 mM and about 0.5M, between about 1 mM and about 100 mM,between about 1 mM and about 50 mM, between about 1 mM and about 10 mM,between about 2 mM and about 8 mM, between about 3 mM and about 7 mM,between about 4 mM and about 6 mM, or between about 4.5 mM and about 5.5mM. The concentration of phosphate ions can generally be anyconcentration, in the range of 0.5 mM and about 2 M. In various furtherembodiments, the phosphate ions are used at a concentration rangingbetween about 0.5 mM and about 2 M, or between about 0.5 mM and about1M, between about 0.5 mM and about 0.5M, between about 0.5 mM and about100 mM, between about 0.5 mM and about 50 mM, between about 0.5 mM andabout 10 mM, between about 0.5 mM and about 7 mM, or between about 1 mMand about 6 mM, between about 1.5 mM and about 5 mM, between about 2 mMand about 4 mM, or between about 2.5 mM and about 3.5 mM. The ratio ofcalcium ions to phosphate ions can generally be any ratio, such as about5:3. In a further embodiment, the oral care product may further comprisefluoride. In various non-limiting embodiments, the fluoride may bepresent in the oral care product at between about between about 50 partsper million (ppm) and about 20,000 ppm, between about 50 ppm and about10,000 ppm, between about 50 ppm and about 5000 ppm, between about 50ppm and about 1000 ppm, between about 50 ppm and about 500 ppm, or about75 ppm and about 400 ppm, between about 100 ppm and about 300 ppm,between about 150 ppm and about 250 ppm, or about 200 ppm. In furtherembodiments, the oral care products comprise at least onebiomineralizing polypeptide, calcium, phosphate, and one or more ofhydrogen peroxide, titanium dioxide, carbamide peroxide,nano-hydroxyapatite particles, and zirconia powder.

Several exemplary embodiments of the present application are summarizedin Table 1 below.

TABLE 1 Product Type Formulation Use area Whitening gel with Fast GelIn-clinic mineralizing Polypeptides Power Bleaching Whitening gel withFast Gel In-clinic mineralizing Polypeptides & Laser Power Bleachinglight Whitening gel with Fast Gel In-clinic mineralizing Polypeptides &Power Bleaching customized tray Whitening gel with Regular Gel At-homemineralizing Polypeptides & Laser fast Bleaching light Whitening gelwith Regular Gel At-home mineralizing Polypeptides & Laser fastBleaching light Whitening gel with Regular Gel At-home mineralizingPolypeptides & Tray fast Bleaching Whitening toothpaste with RegularToothpaste At-home mineralizing Polypeptides Daily Bleaching Whiteningtoothpaste with Mild Toothpaste At-home mineralizing Polypeptides DailyBleaching Whitening toothpaste with Mild Toothpaste At-home mineralizingPolypeptides & 2-3% Daily Bleaching H₂O₂ Whitening toothpaste with MildToothpaste At-home mineralizing Polypeptides & 2-3% Daily Bleachingcarbamide peroxide Whitening Toothpaste with Mild Toothpaste At-homemineralizing Polypeptides Daily Bleaching Whitening solution with MildMouthwash At-home mineralizing Polypeptides Daily Bleaching Whiteningsolution with Mild Mouthwash At-home mineralizing Polypeptides & DailyBleaching chimeric AMPs (separate)

As used in this table, “fast” refers to a rapid single shot whiteningtreatment, “regular” refers to a clinically viable chairside treatmenttime period, and “mild” refers to over the counter consumer products.

All of these aspects/embodiments disclosed herein can be combined withany other aspect/embodiment, unless the context clearly dictatesotherwise.

EXAMPLES Example 1. Polypeptide Synthesis

rM180 amelogenin was created as described previously by Moradian-Oldaket al. (J. Struct. Biol., 2000, 131(1):27-37). The ADPs were synthesizedby standard solid phase peptide synthesis technique on Wang resin usingFmoc chemistry and HBTU activation. CSBio 336s (CSBio, Menlo Park,Calif., USA) automated peptide synthesizer was used for the synthesis.The resulting resin-bound polypeptides were cleaved andside-chain-deprotected using Reagent K (trifluoroaceticacid/thioanisole/H₂O/phenol/ethanedithiol (87.5:5:5:2.5)) and,precipitated by cold ether. The crude polypeptides obtained werepurified by reverse phase high performance liquid chromatography up toa >98% purity (Gemini 10μ C18 110 A column). The masses of the purifiedpolypeptides were checked by mass spectroscopy using a MALDI-TOF massspectrometer (Bruker Daltonics, Billerica, Mass., USA).

Example 2. Experimental In Vitro Procedures of Re-Mineralization Usingthe Peptide Solution on Rat Tooth

Rat teeth (molars) were obtained from sacrificed rats (for example,Sprague Dawley rats, age 3 weeks or older) from animal facility,collected, and stored in 100% alcohol. Prior to the re-mineralizationexperiments the teeth were stored in 1% bleach for 24 hours. The teethdid not have soft tissues or plaque/tartar. The teeth were then dried inan incubator in air at 37° C. for 3-6 hours to produce dry surface. Theroot site and section the crown were removed vertically using a diamondblade. Pictures were taken with camera attached to optical microscope.Exposed dentine sites in the one half of the sectioned tooth were etchedwith 35% phosphoric acid (Ultraetch™) for 60 seconds (to facilitate thestain intake). The etching gel was removed by rinsing the sample withdistilled water for 3 minutes. The staining solution was prepared byboiling 2 g of tea in 100 ml of deionized water for 5 minutes, and thencooled down to room temperature and filtered. The tooth sample wassoaked in the staining solution and incubated at 37° C. at 150 rpm for24 hours. The tooth was removed from the staining solution and sonicatedfor 30 seconds in deionized water (sonic bath) to remove the excessamount of tea particles attached to tooth extrinsically. The one half ofthe sectioned tooth was cut vertically into half again to form twoquarter sections of the tooth. A second set of pictures were taken withcamera attached to optical microscope. One of the quarter pieces wasstored in 24 mM Tris Buffer (pH: 7.4) for control and the other one wasused for in vitro re-mineralization. The portion used for in vitrore-mineralization was submerged into peptide solution (600 μl)containing a mineralization polypeptide in a 48 well plate and incubatedfor 10 minutes at 37° C. After incubation, the mineralized tooth wasplaced in a new well. 400 μl of 9.6 mM CaCl₂ and 400 μl of 5.6 mM KH₂PO₄were added to the well and quickly mixed with a pipetting the solutionup and down. The tooth was incubated in the mineralization solution for16 hours at 37° C. The mineralization polypeptide was SYENSHSQAINVDRT(shADP5; SEQ ID NO:16). The quarter portion of the tooth which had beenexposed to the polypeptide solution, calcium ion source and phosphateion source was observed to be lighter in color than the control

In certain experiments, mineralization was repeated with a freshpolypeptide and mineralization solution for the second round ofre-mineralization.

Example 3. Experimental In Vitro Procedures of Re-Mineralization Usingthe Peptide Solution on Human Tooth

A caries-free human tooth was sterilized in 1% bleach solution for 24hours then removed and vigorously rinsed with deionized water. The rootsite was removed and the crown sectioned vertically using a diamondblade. Pictures were taken with a camera attached to an opticalmicroscope. FIG. 1(a) shows the original tooth before sectioningvertically. FIG. 1(b) shows one half of the original tooth that wassectioned.

The exposed dentine sites were etched with 35% phosphoric acid(Ultraetch™) for 60 seconds (to facilitate the stain intake). Theetching gel was removed by rinsing the sample with distilled water for 3minutes. The staining solution was prepared by boiling 2 g of tea in 100ml of deionized water for 5 minutes, which was then cooled down to roomtemperature and filtered. The tooth was soaked in the staining solutionand incubated at 37° C. at 150 rpm for 24 hours. The tooth was thenremoved from the staining solution and sonicated for 30 seconds indeionized water (sonic bath) to remove the excess amount of teaparticles attached to tooth extrinsically. The sectioned tooth was cutvertically in half again to form two quarter sections of the tooth.FIGS. 2(a) to 2(g) show various sections of the tooth after thestaining. FIG. 2(a) shows the one half of the sectioned tooth after thestaining. Figures (b), (c) and (d) show different views of the leftquarter of the tooth that was sectioned from the tooth. Figures (e), (f)and (g) show different views of the right quarter of the tooth that wassectioned from the tooth. As can be observed from FIGS. 2(a) to 2(g),brown stains from the staining solution were formed on the surface andbelow the surface of the tooth. The left quarter of the tooth was storedin 24 mM Tris Buffer (pH: 7.4) for control and the right quarter of thetooth (test tooth) was used for in vitro re-mineralization. The testtooth was submerged into peptide solution (600 μl) containing amineralization polypeptide in a 48 well plate and incubated for 10minutes at 37° C. After incubation, the tooth was removed and placedinto to a new well. A mineralization solution of 400 μl of 9.6 mM CaCl₂and 400 μl of 5.6 mM KH₂PO₄ was added and quickly mixed by pipetting thesolution up and down. The test tooth was incubated in the mineralizationsolution for 16 hours at 37° C. The mineralization polypeptide used wasSYENSHSQAINVDRT (shADP5; SEQ ID NO:16). FIG. 3 shows the left quarter(control) and the right quarter of the tooth (test tooth). As can beseen in FIG. 3, the right quarter which had been exposed to thepolypeptide solution, calcium ion source and phosphate ion sourceappeared lighter in color than the left quarter (control). In certainexperiments, mineralization was repeated with a fresh polypeptide andmineralization solution for the second round of re-mineralization.

FIG. 4 shows a line profile analysis of a portion of the original tooth(before staining), a portion of the tooth after staining, and a portionof tooth after staining and remineralization. The bar chart in FIG. 4shows the Mean Pixel Intensity (MPI) analyzed using ImageJ™ software onimages of the tooth at before staining, after staining, and afterstaining and remineralization. A zero MPI indicates completely blackwhile one hundred MPI indicates completely white. As can be observedfrom FIG. 4, the portion of the tooth that was stained and hadsubsequently undergone the remineralization treatment showed a higherMPI than the original tooth and the tooth after staining. Therefore, theremineralization treatment with the polypeptide solution and ion sourcesresulted in improved whiteness over the original tooth and the stainedtooth.

Example 4. Mineralization Polypeptide Solution Formulation

An exemplary solution formulation of a mineralization polypeptidedescribed herein consists of Tris buffer pH 7.4, CaCl₂) as the calciumion source, KH₂PO₄ as the phosphate ion source, and the mineralizationpolypeptide.

Example 5. Mineralization Polypeptide Gel Formulation

An exemplary gel formulation of a mineralization polypeptide describedherein is as in follows:

TABLE 2 Ingredient Weight % Solvent: Aqueous 1.6 mM shADP5 polypeptide50 Aqueous 960 mM CaCl₂ Aqueous 576 mM KH₂PO₄ or K₂HPO₄ Preservative:Potassium sorbate 0.5 Thickener/humectant: Propylene glycol (PPG) 3Glycerol (glycerin) 15 Cellulose Gum 1 Sweetener/flavor: sorbitol (60%aqueous solution) 30 Surfactant: simethicone (antifoam) 0.5

Example 6. Mineralization Polypeptide Tablet Formulation

An exemplary tablet formulation of a mineralization polypeptidedescribed herein is as follows:

TABLE 3 Component Weight (mg) Active Ingredient: mineralizationpolypeptide, calcium ions, phosphate ions 300 Diluent: spray-driedlactose (SuperTab ® 11SD) 88 Lubricant: magnesium stearate 6 Glidant:talc 6

Example 7. Solution Mineralization Approach

The root site of an extracted human tooth was removed and the crownportion of the tooth was sectioned vertically into half. A thin layer ofenamel was removed from one half of the sectioned tooth such that thedentin layer is exposed without completely removing the enamel on thetooth. This allowed imaging of the enamel surfaces after themineralization experiment without the problem of surface reflection, andprovides a more parallel surface from which to compare the whitenessresults. The sectioned tooth was fixed onto a glass slide with epoxy.Initial color measurements of each of the four sides of the sectionedtooth were obtained using a Minolta Chroma Meter CR-200 (Konica Minolta,Tokyo, Japan), according to manufacturer instructions, using a whitebackground and a black background. The sectioned tooth was incubated ina mineralization polypeptide solution for 10 minutes at 37° C. and thenin a solution with calcium ions and phosphate ions for 2 hours at 37° C.Color measurements of each of the four sides of the sample were taken asdescribed initially. The sectioned was rinsed with deionized water andthe remineralization process (incubation in polypeptide solution,incubation in solution of ion sources, and rinsing with deionized water)was repeated for a total of 25 rounds. The mineralization polypeptidewas SYENSHSQAINVDRT (shADP5; SEQ ID NO:16). Results of the experimentshowed that whiteness of the tooth enamel improved with each round ofremineralization.

Example 8. Solution Mineralization Approach

Human teeth, donated by the University of Washington School of Dentistry(Seattle, Wash., USA), were selected for their clean, smooth surfaces.The teeth were sterilized in bleach, cut on a diamond blade to removethe root, and halved. A thin layer of the enamel surface was then cut onone of the halves parallel to the previous cut, such that the dentinlayer could be seen without completely removing the enamel on the tooth.This allowed imaging of the enamel surfaces after the mineralizationexperiment without the problem of surface reflection encountered inprevious experiments, in addition to a more parallel surface from whichto compare the brightness or whiteness results. The cut enamel side ofthe tooth sample was then sonicated in DI water and polished on diamondslurry smooth out the surface for imaging. The sample was then cut intoquarters, with the lower half selected for a tetracycline experimentbecause of the even distribution of dentin coloring in the lowerquarters of the tooth sample. The samples were then stained in 10 mg/mLtetracycline solution for five days.

During mineralization, using a 96-well plate, one of the stained halveswas stored in 1200 μL Tris (24 mM) as a negative control, and theexperimental sample was incubated in 150 μL shADP5 (0.8 mM) at 37° C.for 10 minutes. The experimental sample was then rinsed of excesspeptide with deionized water and then stored in 600 μL CaCl₂) (9.6 mM),to which 600 μL monobasic KH₂PO₄ (5.76 mM) was added. The well platecontaining both the control and experimental samples was then stored at37° C. for about 24 hours for mineralization to occur on the treatedstained tooth sample.

The remineralization process (incubation in polypeptide solution,incubation in solution of ion sources, and rinsing with deionized water)was repeated on the experimental sample for a total of 27 rounds. After27 rounds of mineralization on the experimental sample, the tooth wasinspected for changes in brightness qualitatively. The stained negativecontrol was polished to expose the original enamel stain in order tocompare the whitening effect of the peptide mineralization. FIG. 5 showspictures of the tooth before staining, a side-by-side comparison of thestained negative control sample with the experimental sample havingthree rounds of remineralization, a side-by-side comparison of thestained negative control sample with the experimental sample having fourrounds of remineralization, a side-by-side comparison of the stainednegative control sample with the experimental sample having five roundsof remineralization, and a side-by-side comparison of the stainednegative control sample with the experimental sample having seven roundsof remineralization. FIG. 6 shows a line profile analysis of the toothbefore staining (Bar A), the 3-rounds remineralized tooth (Bar B), the4-rounds remineralized tooth (Bar C), the 5-rounds remineralized tooth(Bar D), and the 7-rounds remineralized tooth (Bar E). The bar chart inFIG. 6 shows the Mean Pixel Intensity (MPI) of the tooth at the variousstages.

The results are as follows, showing effective whitening from use of thebiomineralization peptide:

TABLE 4 Assay Mean Pixel Intensity Bar in FIG. 6 No staining or 70.22 ±1.57 A remineralization After 3 rounds 69.47 ± 2.96 B After 4 rounds70.11 ± 2.92 C After 5 rounds 70.79 ± 1.96 D After 7 rounds 74.47 ± 2.13E

As can be observed from bars (B) to (E) in FIG. 6 and the mean pixelintensity values in the table above, whiteness of the stained toothimproves with repeated rounds of remineralization.

Example 9. Biomimetic Tooth Repair: Amelogenin-Derived Peptide Permit InVitro Remineralization of Human Enamel

White spot lesions (WSL) and incipient caries on enamel surfaces are theearliest clinical outcomes for demineralization and caries. If leftuntreated, the caries can progress and may cause complex restorativeprocedures or even tooth extraction which destroys soft and hard tissuearchitecture as a consequence of connective tissue and bone loss.Current clinical practices are insufficient in treating dental caries. Along-standing practical challenge associated with demineralizationrelated to dental diseases is incorporating a functional mineralmicrolayer which is fully integrated into the molecular structure of thetooth in repairing damaged enamel. This study demonstrated that smallpeptide domains derived from native protein amelogenin can be utilizedto construct mineral layer on damaged human enamel in vitro. Six groupswere prepared to carry out remineralization on artificially createdlesions on enamel: (1) No treatment, (2) Ca²⁺ and PO₄ ³⁻ only, (3) 1100ppm fluoride (F), (4) 20,000 ppm F, (5) 1100 ppm F and peptide, and (6)Peptide alone. While the 1100 ppm F sample (indicative of common Fcontent of toothpaste for homecare) did not deliver F to the thinlydeposited mineral layer, high F test sample (indicative of clinicalvarnish treatment) formed mainly CaF₂ nanoparticles on the surface.Fluoride, however, was deposited in the presence of the peptide whichalso formed a thin mineral layer, which was partially crystallized asfluorapatite. Among the test groups, only the peptide-alone sampleresulted in remineralization of fairly thick (10 μm) dense mineralizedlayer containing plate-like HAp resembling the structure of the healthyenamel. The newly formed mineralized layer showed integration with theunderlying enamel as evident by cross-sectional SEM imaging. Theapproach has potential utility in future clinical implementation as anew, biomimetic remineralization treatment in dental health care.

Dental caries is one of the major public health problems and it is ahighly prevalent disease among the global population. Incipient cariesand white spot lesions (WSL) as well as hypersensitivity, are theearliest clinical evidence of enamel demineralization and dental caries.Dental caries occurs when tooth enamel is exposed to acid produced bycariogenic bacteria. As a result, acid diffuses into surface enamel anddissolves hydroxyapatite (HAp) mineral. Due to its non-regenerativenature, enamel is unable to heal and repair itselfpost-demineralization.

Traditionally, fluoride (F) has been used as the key agent in preventionof caries. Fluoride functions primarily via topical mechanisms. It isbelieved that fluoride forms a thin layer of new but harder mineral,namely fluorapatite (FAp) which is incorporated into the existing HApmineral on the tooth surface. There is a trend of enhancing theremineralization effect of fluoride with calcium and phosphatesupplementation in high risk individuals. Although controversial, theuse of fluoride products remains the primary treatment modality forcaries prevention and remineralization, with major limitations regardingthe efficacy of these products for the reversal or prevention of dentalcaries. Fluoride delivery systems, therefore, are not sufficient toovercome the high caries risk especially in younger and elderlypopulation.

Although dental caries is a preventable infectious disease, oral healthpromotion and prevention can fail due to many factors. The advancedcavitation of the carious lesion necessitates restoring the tooth withmaterials such as metals, composite resins and ceramics to replace thelost enamel or, even, dentin. However modern dental materials to repaircavitated carious lesions are not compatible with biological tissues atthe lesion/restorative material interface mainly because of theirphysical (crystallography and morphology) and chemical differences(elemental compositions and phases) compared to the natural toothstructure. Although treatment of early caries lesions by the applicationof various types of nano-sized HAp or CaCO₃ with or without F hasreceived considerable attention, their clinical validation is stilllacking. Low solubility of the calcium phosphates, particularly in thepresence of fluoride ions is the main difficulty with the clinicalapplication of remineralization. No clinical remineralization system hasemerged to promote biomimetic enamel subsurface remineralization invivo.

Incorporating a functional and biomimetic mineral layer to the molecularstructure of the tooth to repair damaged enamel tissue has been a longstanding challenge. A better understanding of peptide-guidedremineralization on human tooth and, therefore, the ability to controlthe mineral layer properties, with no, low or high-F content, hasenormous clinical implications to restore enamel and other dental hardtissues. As a major step towards this overarching goal, the objective ofthis study has been to develop an in vitro, cell-free, naturalremineralization model on artificially induced enamel lesions Because ofthese unique biomineralization characteristic and its short and simplesequence, ADPS has been used in this research as the active ingredientin solution for the formation of mineralized layer for the purpose ofrepairing artificially formed defects on the surface of enamel. The workpresented herein could eventually form the foundation of developingclinical treatments for the restoration of early stage cavities, e.g.,incipient caries, white spot lesions and hypersensitivity.

Materials and Methods Design of Amelogenin-Derived Peptides, ADPs

The ADPS peptide was generated using a procedure that was developed fordesigning protein derived peptides as described previously. Briefly,7-AA and 12-AA long hydroxyapatite binding peptides (HABPs) wereexperimentally selected using the c7c- and 12-phage libraries (NewEngland BioLabs Inc., Ipswich, Mass., USA), respectively. Next, the 180amino acid-long human amelogenin protein (rM180) was divided into 7-AAand 12-AA long segments and each segment was compared with all of the155 experimentally selected HABPs. The regions with high similarityscores from 7-AA and 12-AA long segments were overlapped and thosecoinciding high similarity regions were chosen as the putative strongbinding regions. These computationally determined high similarityregions were then refined to design the ADPs by protein structureprediction, Ca²⁺ ion-binding domain predictions, and meta-functionalsignature analyses. Two separate assays were developed to characterizethe ADPs: Solid binding affinity to HAp and mineralization in aqueoussolution. The prediction in the binding assay was 100% while thebiomineralization assay resulted in some surprises. Relevant to thisstudy, although ADPS is one of the weakest binding peptides, it has thefastest mineralization kinetics, very close to that of full-lengthamelogenin. Because of this property, ADPS has been used for developingmineralization on human teeth. One more aspect of ADPS is that its fulllength, 25 AA, is difficult to solubilize in water. For this, weeliminated the six amino acids from the amino end and alanine from thecarboxyl end, while keeping the charged amino acids intact that arethought to initiate mineralization. The originally designed ADPS wasgenerated from mouse amelogenin; for shADP5 K in the sequence wasreplaced by N, where “s” stands for “short” and “h” for “human”. TheshADP5 peptide was synthesized by solid phase peptide synthesis CSBio336s (CSBio, Menlo Park, Calif., USA) automated peptide synthesizer andpurified by reverse phase high performance liquid chromatography. Themasses of the purified peptide and its purity were confirmed by usingMALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, Mass., USA).Molecular characteristics of the shADP5 peptide used in this work arelisted in Table 8.

Sample Preparation and Test Groups:

Extracted teeth with no visible white spot lesions, caries, or any otherkind of restorations were collected from dental clinics around the KingCounty area (WA, USA) and disinfected in 10% aqueous bleach solutions.Prior to the experiments, the teeth were cleaned to remove visibleblood, gross debris and soft connective tissue using a dental scalerunder a light microscope.

Formation of Artificial Lesions

Enamel was demineralized to create artificial lesions to mimic whitespot lesions and/or incipient caries. The teeth were covered by lacquerleaving a 4-mm wide square window in the enamel close to cemento-enameljunction. The exposed areas of 4×4 mm enamel were treated with acocktail of acetic acid/CaCl₂/KH₂PO₄ for 2 weeks to establish up to 200μm deep artificially created non-cavitated white spot lesions (WSL).Samples were then divided into control and test groups. Test groups weretreated either with peptide, fluoride, or combination of both (Table 5).

TABLE 5 Experimental Test Groups and Mineralization Treatments. # ofTest Groups Treatment samples Group-1: No Treatment 5 (Negative Control)Group-2: (Ca²⁺ and PO₄ ³⁻ 4.8 mM (Ca²⁺/2.88 mM PO₄ ³⁻, 1 5 Only) hourGroup-3: 1100 ppm F (common fluoridated 5 (Low concentration F)toothpaste concentrations), 4.8 mM Ca²⁺/2.88 mM PO₄ ³⁻, 1 hour Group-4:20,000 ppm F (dental varnish 5 (High concentration F) concentration),4.8 mM Ca²⁺/2.88 mM PO₄ ³⁻, 1 hour Group-5: 1. 0.8 mM Peptide. 10minutes 5 (shADP5 with low 2. 1100 ppm F + 4.8 mM Ca²⁺/2.88concentration F)  mM PO₄ ³⁻, 1 hour Group-6: 1. 0.8 mM Peptide, 10minutes 5 (shADP5) 2. 4.8 mM Ca²⁺/2.88 mM PO₄ ³⁻,  1 hour

Peptide Design and Synthesis

The peptide shADP5, shortened ADPS, was generated using a procedure thatwas developed for designing protein-derived peptides, as describedpreviously³¹ (see also short synopsis of the procedure in SupportingInformation). The peptide (Table 6) was synthesized by using anautomated solid-phase synthesizer (CS336X; CS-Bio, Menlo Park, Calif.,USA) through Fmoc-chemistry. In this procedure, in the reaction vessel,the Wang resin (Novabiochem, West Chester, Pa., USA), was treated with20% piperidine in DMF to remove the preloaded Fmoc group. Next, theincoming side chain protected amino acid was activated with HBTU(Sigma-Aldrich, St Louis, Mo., USA) in dimethylformamide (DMF,Sigma-Aldrich, St Louis, Mo., USA) and then transferred into the vesselwhere it was incubated with the resin for 45 min. After washing theresin with DMF, this protocol was applied for the addition of each ofthe next amino acids and synthesis reaction was monitored byUV-absorbance at 301 nm. Following synthesis, the resulting resin-boundpeptides were cleaved and the side-chain de-protected using reagent-K[TFA/thioanisole/H₂O/phenol/ethanedithiol (87.5:5:5:2.5), Sigma-Aldrich,St Louis, Mo., USA] and precipitated by cold ether. Crude peptides werepurified by RP-HPLC with up to >98% purity (Gemini 10u C18 110 Acolumn). The sequence of the peptides was confirmed by a MALDI-TOF massspectrometry with reflectron (RETOF-MS) on an Autoflex II (BrukerDaltonics, Billerica, Mass., USA).

TABLE 6 Molecular characteristics of the peptide shADP5. Net PeptideSequence MW pI G.R.A.V.Y. Charge shADP S Y

NS

SQ 1720.7 5.30 −1.273 −1 AI N V

T* (SEQ ID NO: 16) *Bold font AAs are polar, uncharged residues;underlined residues are hydrophobic; italicized residues are chargedresidues G.R.A.V.Y. is grand average of hydropathy (see web sitegravy-calculator.de/)

Re-Mineralization Protocol

Prior to remineralization, samples requiring peptide treatments (Groups5 & 6) were incubated in 50 μL of 0.8 mM peptide dissolved in 50 mM TrisBuffer Solution (TBS) (pH:7.4) for 10 minutes at 37° C. Next, treatmentsamples were exposed to 50 mM TBS containing Ca²⁺/PO₄ ³⁻ (Groups 2, 5)or Ca²⁺/PO₄ ³⁻/F⁻ (Groups 3, 4, 6) at concentrations as listed in Table5 for 1 hour at 37° C., then rinsed with de-ionized (DI) water, dried byforced air and stored at room temperature until characterization.

Sample Characterization by SEM and EDXS Analyses—Imaging and ElementalComposition:

After re-mineralization experiments were completed, secondary electronimaging (SEI) in the scanning electron microscope (SEM) was used tocharacterize the surface morphology and to show the thickness of newlyformed mineral layer in cross-sections where applicable. Specimenpreparation for SEM involved cutting a notch on the back side using alow speed saw (IsoMet™, Buehler, Lake Bluff, Ill., USA) before they weresubjected to WSL formation and remineralization as described above.After remineralization step was completed, specimens were rinsed with DIwater, air dried gently (<5 PSI), then carefully fractured into 2 piecesalong the notch. One of the fractured pieces was mounted on a SEM stubwith the mineralizing surface facing up for imaging the surfacemorphology and second piece was mounted with the cross-section facing upto show the thickness of mineral layer. Mounted specimens were thenstored in vacuum for at least 2 hours to remove residual moisture whichwere then sputter coated with 5-nm thick platinum (SPI-Sputter ModuleCoater, SPI Supplies, West Chester, Pa., USA). SEM characterization wasperformed using an FEI Sirion microscope (Sirion, FEI, Hillboro, Oreg.,USA) operating at 10 kV acceleration voltage. The chemical compositionwas measured by an onboard energy dispersive X-ray spectroscopy (EDXS)system (X-Max^(N) Si drift detector with AZtecEnergy software package,Oxford Instruments, Abingdon, Oxfordshire, UK). The measurements foreach group were pooled from 5 specimens per group. The average valuesand standard deviations were calculated and expressed as themean±standard error.

Structural Characterization by Transmission Microscopy (TEM)

After remineralization steps were completed, TEM samples were collectedby carefully shaving off the top most surface of the remineralized layerfrom the artificially created white spot lesion using a clean razorblade. The shaved particles were suspended in 100% ethanol, and thesuspension was drop-casted onto a carbon coated TEM grid, which was thenvacuum dried before TEM characterization. TEM bright field imaging (BF)imaging and selected areas diffraction were carried out using an FEITecnai (FEI, Hillboro, Oreg., USA) operating at 200 keV.

Mechanical Properties Characterization

Similar to SEM specimen preparation, tooth samples were notched from theback of tooth before remineralization, then fractured along the notch.The specimens were then mounted in a room temperature-cure epoxy, andthe cross-section of the fracture was polished to 0.1 μm finish usingdiamond lapping films (Allied High Tech Products Inc., Rancho Dominguez,Calif., USA). Nanoindentation measurements were made using aTriboindentor nanoindentation system (Hysitron Inc., Minneapolis, Minn.,USA) in air. Hardness (H) and elastic modulus (E_(r)) were determined bythe software accompanying the nanoindentation unit.⁴⁰⁻⁴² In order toobtain the values that were not indentation volume dependent, maximumindentation depth for all measurements kept at 120±10 nm. All reported Hand E_(r) values were averaged over 20 measurements.

In addition to nanoindenation measurements, microhardness was alsoperformed on the surface to quantitatively assess the mechanicalproperties of the mineral layers with larger areas and volumes includingthe underlying enamel as a composite. Vicker's micorhardness wasperformed on at room temperature using Vicker's indenter on a WilsonHardness Tukon 1202 microhardness tester at 10 kg applied load (IllinoisTool Works, Lake Bluff, Ill.). At least 20 measurements per group wererecorded for obtaining an average and statistical analysis.

Results

The incubation of samples in demineralization cocktail exposed enamelrods on the surface of the samples before the re-mineralizationtreatment was undertaken, as shown in Group 1-Negative Control (FIG.8a-b ). Elemental chemical analysis of the surface by EDXS gives a ratioof Ca²⁺/PO₄ ³⁻ 1.56±0.12 (FIG. 8c ). As seen in the cross-sectional viewof (FIG. 8d ), well aligned enamel rods of ˜3 μm diameter extend to theexposed surface where they display HAp crystallites constituting therods. After 1-hour of exposure to Ca²⁺/PO₄ ³⁻ solution, no substantialre-mineralization was observed on the samples in Group 2. Consideringthat the imprints of enamel rod remained visible as shallow depressionson the enamel surface (FIG. 8e-f ) any possible deposit of solidmaterial, possibly the result of Ca²⁺ and PO₄ ³⁻ ions reacting to forman amorphous deposit, remained extremely thin. In fact a very thin (<1μm) layer is barely visible in the SEM image of the cross-sectionedsample shown in FIG. 8h . Elemental analysis of the surface by EDXSgives a ratio of Ca²⁺/PO₄ ³⁻ 1.45, possibly indicating a mixed mineralcomposition (FIG. 8g ; also see Table 7).

TABLE 7 Elemental composition analyses of the remineralization testgroups by EDXS. Test Remineralized Layer Possible Mineral Group Ca/PCa/F Ca/O Formed 1. Control 1.56 ± 0.12  — 0.51 ± 0.11 Only HAP 2. Ionsonly 1.45 ± 00.04 — 0.28 ± 0.02 Amorphous Ca—P transition phase 3. Low F1.69 ± 0.05  36.99 ± 02.9 0.74 ± 0.15 Ca—P—F transition phase + CaF₂ 4.High F 5.62 ± 0.96   0.49 ± 0.11 1.01 ± 0.16 Mainly CaF₂ 5. Low F + 1.60± 00.11  8.74 ± 0.78 0.34 ± 0.18 FAP + some   Peptide CaF₂ 6. Peptideonly 1.56 ± 00.12 — 0.56 ± 0.13 Only HAP

In Group 3 (low concentration fluoride), 1100 ppm F was applied in thepresence of Ca²⁺ and PO₄ ³⁻ ions. The concentration of 1100 ppm fluoridecorresponds to the concentration of the most commonly used toothpasteavailable over the counter for daily home care.⁵⁻⁷ The analysis of theSEM images recorded from the surface suggests non-uniformly depositedlayer with a fine (<1 μm) roughness compared to the surface formedwithout the fluoride in Group 3 (FIG. 9a-b ). A detailed analysis of thesurface structure, e.g., at higher magnification image in FIG. 9b ,reveals fine nanoparticles of diameter 20-50 nm. The cross-sectionedsamples reveal a new layer with a thickness of about 1 μm covering thesurface of enamel in the lesion (FIG. 9d ). The elemental compositionanalysis from the surface revealed prominent peaks of O_(Kα), F_(Kα),and Ca_(Lα), as well as a small peak corresponding to F_(Kα). Ca/F ratiogives a values of more than 30 while the Ca/P ratio is close to 1.70(FIG. 9c ).

In Group 4 (high concentration fluoride), 20,000 ppm fluoride(concentrations of most commonly used dental varnishes) applied withCa²⁺ and PO₄ ³⁻ ions. The analysis of the SEM images recorded from thistreatment displayed significantly different surface topography,structures, and elemental composition as compared to the samples in theprevious groups of non-fluoride or low concentration F treatment.Although at low magnifications (FIG. 9e ) the surface appears fairlysmooth, higher magnification (FIG. 90 revealed small spherical particlesof 100-200 nm diameter covering the overall surface (indicated by arrowin FIG. 90. The SEM secondary electron images recorded from thecross-sectioned samples reveal an about a micrometer-thick new layer onthe surface of the teeth (FIG. 9h ). The EDXS spectra received from thesurface gives a high concentration of F_(Kα) peak, the most prominentamong all the peaks in the spectra from this group of samples (FIG. 9g). The quantitative analysis of the spectra from the samples prepared inthis group exhibited the C/F ratio of 0.49 (Table 7).

In group 5 (the peptide with low concentration fluoride) shADP5 wasapplied with 1100 ppm fluoride along with Ca²⁺ and PO₄ ³⁻. The surfacemicrostructure of the samples carried out by SEM showed fairly smoothsurface with about 1-2 μm thickness (FIG. 10a-d ). Enamel rod imprintsremained visible in the lower magnification image (FIG. 10a ). Highermagnification image of the sample surface, however, exhibits twodifferent surface morphologies (see insets in FIG. 10b ); somewhatloosely deposited nanoparticles of 50-100 nm diameter and densestructure composed of rod-like nanoparticles of few tens of nanometersin diameters with the diameter/length aspect ratio of 1/5. Elementalanalysis of the samples from this group revealed fairly noticeableF_(Kα) peak in addition to highly prominent Ca_(Kα) and P_(Kα) peakswith the elemental ratio of Ca/F, 8.7 (FIG. 10c ).

In the Group 6 (shADP5+Ca²⁺/PO₄ ³⁻), the SEM images in FIG. 10e-f give acontinuous layer of plate-like crystals growing from the surface of theunderlying enamel lesion when the surface is exposed to aqueous peptideplus Ca²⁺/PO₄ ³⁻. Compared to the negative control (Group-1) or lowconcentration fluoride treatment (Group-3), the enamel rod imprints inthe face-on images are no longer visible, indicating that the newmineral layer is thick enough to mask the exposed enamel rods (FIG.10e-f ). The cross-section image in FIG. 10h shows a 10 μm thickcontinuous remineralized layer with fairly smooth surface topography.Elemental analysis of the samples from this group revealed prominentCa_(Kα) and P_(Kα) peaks with a ratio of 1.54±0.12; this is close toideal ionic ratio of 1.6 in HAp composition (FIG. 10g ; Table 7).

To further analyze the structural characteristics of the mineral layersin experimental group, imaging and diffraction analyses were carried outon samples by using transmission electron microscopy. The TEM sampleswere prepared by gently shaving fragments off the surface of toothspecimens. Group 1, which received no remineralization treatment, enamelfragments were analyzed. As shown in FIG. 11a-c , textured elongated HApcrystals of 30-50 nm were encountered, typical of those in enamel rodsin healthy enamel tissue. In the case of high concentration F treatment(group 4), generally round particles CaF₂ in the range of 100-250 nm indiameters were observed (FIG. 11d-f ). On the peptide treatment group(group 6), large particles (in the shown projection) of HAp were found(FIG. 11g-i ) possibly corresponding to plate-shape mineral particles.It is noted that groups 2, 3 and 5, had structural characteristicssimilar to those of group 1 were discovered (data no shown here butdisplayed in the SI section). It should be noted, however, that in allcases, it was challenging, but not impossible (as demonstrated in FIG.11 above) to differentiate the newly formed crystallites from those HApcrystallites in the underlying enamel.

Mechanical properties of the mineralized layers were determined from twotests. First, the microhardness test was carried out using a Vicker'sindenter loading on the mineralized tooth surface. The hardness for thenegative control group (Group 1), i.e. no treatment was 130.1±10.4 HV10.This was the baseline figure representing microhardness of the surfaceof bare artificially created WSL which other experimental groupscompared against. As the reference, the microhardness tests were alsoconducted on healthy enamel and healthy dentin, away from themineralized surface, and displayed in Table 8. As shown in Table 8,values for groups 2 to 5 ranged between 129.6±14.9 HV10 to 133.7±12.9HV10. Student's t-test between group 1 and each of these groups revealedno statistically significant difference (p>0.05). Microhardness of group6 had slightly higher average value of 140.6+11.3 HV10. Student's t-testagainst group 1 revealed significant difference with p<0.01. The resultsindicate that the microhardness values of group 6 as well as the rest ofexperimental groups fell between that of enamel and dentin.

TABLE 8 Vicker's microhardness of all experimental groups, n ≥ 20.Hardness Test Groups (HV10, MPa) Group 1 (Negative Control) 130.1 +/−10.4 Group 2 (Ca/PO₄ Only) 132.5 +/− 14.9 Group 3 (Low Conc. F) 129.6+/− 13.8 Group 4 (High Conc. F) 131.9 +/− 13.3 Group 5 (shADP5 + LowConc. F) 133.7 +/− 12.9 Group 6 (shADP5) 140.6 +/− 11.3 Healthy Enamel290.3 +/− 36.4 Healthy Dentin 63.3 +/− 3.0

Second mechanical tests were conducted at the nanometer-scale bynanoindentation which not only provides hardness (H) values but alsoelastic modulus (E_(r)), and the test could be carried out spatiallyselected regions as the test facilitates scanned surface images.Therefore, nanoindentation tests of all the experimental groups wereconducted in cross-sectioned geometry, i.e., indentor direction isparallel to the surface (as opposed to vertical in microhardness tests).The results are tabulated in FIG. 12. Similar to the trend shown in themicrohardness data, significant differences were not encountered betweengroup 1 (no treatment negative control) and each of groups 2 through 5in both hardness and reduced elastic modulus, p>0.05 in all cases.However, observed here again, the average hardness and elastic modulusfor group 6 were higher than that of no treatment group 1 with hardnessof 2.23±0.23 GPa vs. 2.10±0.36 GPa, p=0.02 and elastic modulus of58.6±4.7 GPa vs. 55.1±4.3 GPa, p=0.02. Not surprisingly the healthyenamel and dentin had respectively higher and lower values of bothhardness and elastic moduli compared to the experimental groupsinvolving remineralization. In conclusion, the mechanical properties (Hand E) are higher than those of dentin, but lower than the healthyenamel.

DISCUSSION

A natural, cell-free, biomimetic model was developed to re-mineralizeartificially induced lesions on human enamel using a 15-amino acid longamelogenin-derived peptide, shADP5, along with properly tuned ionicconcentrations of Ca²⁺/PO₄ ³⁻ in vitro in the presence and absence oflower and higher fluoride content which were chosen based on the valuesin the frequently used present dental treatments.

There are drastic differences in the surface characteristics among the 6groups. First of all, the surface of the artificially demineralizedenamel displayed enamel rods, which appeared up to 3-μm diameterdepressions exposed on the surface (FIG. 8a-b and FIG. 11a-c ). Otherprominent feature on the surface was the fine structure of theindividual rod-shaped HAp crystallites of a few tens of nm thickness ofwhich make up the enamel rods. However, variations in the microstructureof the artificially demineralized enamel were observed on differenttooth samples. As a result of demineralization, the enamel rods of somespecimens were more prominent than other samples due to individualdifferences in tooth structure and cross-section orientation whichexplains the differences in their apparent local morphology andcomposition on the surfaces of the teeth samples.

When ionic precursors are used alone (Group 2), this resulted in a thin(<1 μm) layer with a highly porous morphology. The composition was offstoichiometric, i.e., Ca/P ratio of <1.5 which might be due to theformation of calcium-phosphate transition phases (Table 7 and Table 9).We next studied the effect of fluoride on mineral formation which wasexamined under two different fluoride ion conditions, 1100 ppm in Group3 and 20,000 ppm in Group 4, which were specifically chosen to mimic thefluoride concentrations of everyday tooth paste and clinical fluoridevarnish used in the clinic, respectively. Close examination of thesurface structures of the teeth in these two treatments revealeddifferent morphologies. First of all, fluoride treatments resulted inaggregates of nanoparticles in Group 3, and a thin mineralized layer;about 1 μm in Group 4. The layers were composed of nanoparticles whichwere about an order of magnitude smaller in Group 3 than in Group 4samples, 20 nm versus 200 nm, respectively. The application of fluoridein dental care products primarily focus on remineralization that isaided by fluoride or incorporation of fluoride into the existing HApstructure, desirably forming fluorapatite (FAp). In this respect, theresults of the elemental analyses obtained from the fluoride-treatedsurfaces are quite intriguing. The Group 3 samples, with low-F,presented hardly any F peaks in the EDXS spectra, giving Ca/F ratio ofalmost 40. The elemental composition analysis of the sample surfacedisplayed relatively high relative peaks of O_(Kα), P_(Kα), and Ca_(Lα)as well as a small peak corresponding to F_(Kα) (Na_(Kα) and Cl_(Lα)peaks are from the treatment solutions). Possible sources of the low Fconcentration might be explained either due to the formation of verythin layer of the deposited solid on the surface resulting in thesignals originating from the underlying healthy enamel as measured inthe majority of EDXS or due to the very low amount of F incorporatedinto the newly formed surface layer. The lack of sufficient elementalcomposition of F in the newly formed layer may imply that majority of Fwas not delivered to the desired mineralization site on the enamelsurface in the peptide-free samples. The presence of nanoparticlesdeposited on the surface forming a thin layer, therefore, may be due toF reacting with excess Na in the buffer solution forming NaF.

In Group 4, the results from face-on and edge-on images illustrated thatthe new layer is predominantly composed of aggregation of sphericalnanoparticles. Considering the ideal Ca/F ratio of 0.5 in CaF₂ (Table 9,and FIG. 11d-f TEM results), the spherical particles might be CaF₂mineral. Another major difference in this group was the value ofCa_(Kα)/P_(Kα) ratio, which was more than 5.0. Even considering thatsome of the Ca ions might be confined in CaF₂, this ratio stillindicates unusually high concentration of Ca trapped in the newly formedlayer on the enamel surface. CaF₂ is a highly stable compound that couldform under the experimental conditions of this study. Instead of theintended apatite compound, the mineral formed in this group was mostlikely calcium fluoride. Although the ideal ratio of Ca/P is 1.6 for HAp(Table 3), neither of the F-alone samples revealed such concentrationratio. The significance of this result mean that F-alone was notdelivered to the tooth surface and, as a consequence, was notincorporated into the enamel or remineralized structure on the surfaceunder the experimental conditions. The question remains therefore thatduring the clinical and everyday applications of fluoride whether thesame formation takes place, i.e., CaF₂ materialization instead ofincorporation of F into the HAp mineral within the tooth structure.

In the last two groups of test samples (Group 5 and 6) shADP5 peptidewas used as part of the precursors during the remineralizationexperiments. In Group 5, remineralizing peptide shADP5 was applied inthe presence of 1100 ppm fluoride. Under low concentration of F, thespherical particles had a tendency to form island aggregates as opposedto being widely disseminated on the demineralized enamel surfacecompared to the same fluoride concentration without shADP5 in Group 3.Enamel rod imprints remained visible in the lower magnification image(FIG. 10a-b ) although these are less prominent than those ofno-treatment samples (FIG. 8a-b ). The resulting mineralized structurepresented two morphologies: the first one was clusters of 50-100 nmdiameter spherical nanoparticles accreted non-uniformly on the surface;and the second structure was primarily composed of highly densenanorods. There was considerably more prominent F peak compared to theno peptide samples, with an overall Ca/F ratio of 8.74±0.78. Thisexplains that there was considerably more F in the mineral formed withthe peptide and low fluoride treatment compared to the low fluorideconcentration only, (Ca/F=36.99±2.9). If the presence of FAp isconsidered, i.e., corresponding to the dense nanorods, the ideal ratioof Ca/F is 5.0 (Table 7), then the rest of the fluoride in the minerallayer could be accounted for the formation of NaF nanoparticles.Considering that the observed Ca/P ratio reflects either HAp or FApstoichiometry (Table 7), the conclusion could be drawn here that the newmineral was formed on the teeth surfaces by partially incorporating F inthe presence of peptide. The presence of F could be explained in, atleast, two ways; either the fluoride was incorporated into the newlyforming HAp mineral replacing OH partially or both HAp and FAp wereformed on the surface. In both cases, the effect of peptide appears tobe necessary to incorporate fluoride into the structure since in theabsence of shADP5, very little or no F was found in the remineralizedlayer. This may mean that the materials used in the current treatments,e.g., pastes, gels, varnish, and solutions, would contain peptide as ameans to effectively mineralize HAp and but also as a carrier for F tothe newly mineralized layer and effectively incorporating into it.

In Group-6, in addition to calcium and phosphate precursors, shADP5peptide was included in the solution. In this case, a thick (>10 μm)remineralized layer formed on the surface composed of plate-like crystalmorphology. Considering that this crystal habit is specific to HAp amongthe calcium phosphate polymorphs and the observed Ca/P ratio is 1.56which is close the ideal HAp composition, the shADP5 peptide was capableto form a newly mineralized layer composed of HAp crystallites (FIG.11g-i ), demonstrating the remineralizing capability of this peptide aswell as layer formation covering the damaged enamel on the human toothsurface. Measuring microhardness of each experimental group on thesurface was a way to assess the mechanical properties of the mineralizedsurface of the experimental groups versus no treatment negative controlgroup in a loading direction relevant to functional dentition loadingwhile using the lowest possible Vicker's indentation load maximize thecontribution from the mineralization layer. Even with this lowest load,however, the Vicker's indentor was likely to be penetrating through thethin mineralized layers in groups 2-5; this explains that there are nomeasurable differences discernable among these groups and theno-treatment negative control group. This explanation is furthersupported by the SEM observations which showed mineral layers in groups2 through 5 were in the order of a few μm (perhaps as thin as 1 μm), andwith some discontinuity. Because the mineral layer was much thicker ingroup 6, not only a real difference in microhardness was detectedcompared to the negative control group, it is revealed that it wassignificantly harder, though only slightly. The small mineral layerthickness can also explain no detectable difference by nanoindentationin cross-section between group 1 and groups 2-5. It is indeedchallenging to discern the mineral layer and underlying WSL in groups 2to 5 when performing the measurements. The higher Vicker's hardness inthe peptide mineralization group (group 6) compared well to the controlgroup (group 1). Combined with the higher hardness in cross-sectionorientation, measured by nanoindentation, suggests that the new layersformed in group 6 samples were harder than the underlying artificiallycreated WSL enamel.

CONCLUSIONS

The present in vitro study demonstrated that crystalline mineral layercan be formed on artificially created lesions on human enamel in thepresence of Ca²⁺ and PO₄ ³⁻ ions under physiologically viable conditionsby using shADP5, a 15-AA long amelogenin-derived peptide. This studyalso showed that the presence of biomineralizing peptide, shADP5, allowsthe delivery and incorporation of fluoride ions into the remineralizedlayer even at low F concentrations, providing an opportunity for dentalhealth products to incorporate both elements in some clinical oreveryday utility settings.

Supplemental Data

Relevant Compounds among Ca, P, F and O

A set of possible compounds that may form under the experimentalconditions discussed here upon the mineralization are listed in Table 9below, with relevant elemental concentration ratios. For example, Ca/Pratio for HAp is 5/3=1.6 and for FAp, Ca/P is 5/1=5. In some cases,e.g., ions only test group, (Group 2), since most of theremineralization products on the surface of enamel are a result ofdeposition, instead of remineralization, a variety of transition Calciumphosphate solids may form, all in their amorphous, and, hencenon-equilibrium states. In other words, these are kinetically trappedphases rather than being thermodynamically stable compounds.

TABLE 9 Relevant Compounds of Ca, P, F, O and Na. Compound. Formula Ca:PCa:F Ca:O Na:F Na:P Hydroxyapatite, HAP Ca₅(PO₄)₃(OH) 1.60 — 0.38 — —Octacalcium Phosphate, OCP Ca₈H₂(PO₄)₆•5H₂O 1.33 — 0.28 — —Fluorapatite, FAP Ca₅(PO₄)₂F 1.67 5.00 0.42 — — Monocalcium Phosphate,MCP Ca(H₂PO₄)₂ 0.50 — 0.13 — — Dicalcium Phosphate, DCP CaHPO₄ 1.00 —0.25 — — Tricalcium Phosphate, TCP Ca₃(PO₄)₂ 1.50 — 0.38 — — CalciumFluoride, CF CaF₂ 0.00 0.50 — — — Sodium Fluoride, NaF NaF — — — 1.000.00 Na-monoflourophosphate, MFP Na₂PO₄F — — — 2.0 2.00

Supplemental TEM Data

Structural and phase characterization of the samples from all groupswere undertaken by transmission electron microscopy imaging anddiffraction analysis. This is because x-ray diffraction characterizationrequires large enough volume with 10s of μm mass thickness and largerspatial sampling, 100s of μms or larger areas. Since the mineralizedlayer is thin, a few μms in all other group samples to 10 μm or so ingroup 6 samples using the peptide, TEM has been used to provide the mostcritical data, diffraction and phase identification. The TEM sampleswere prepared by gently shaving fragments off the surface of toothspecimens onto carbon-coated TEM grid to prepare and ensure electrontransparent sections. All of the TEM data is included in FIG. 13 belowfor completeness (while only 1^(st), 4^(th) and 6^(th) columns of datawere presented in FIG. 11).

While the no treatment sample (a-c) shows plate-like crystals of HApthat probably constitute the enamel rod, typical of the sound enameltissue, the sample prepared with only the Ca and PO4 ions show onlyeither small particles or structures that possibly correspond to theunderlying enamel in the artificially lesioned surface. Low F and high Fsample display clusters of nanoscale particles and larger particles with100 nm diameter or larger, respectively. These results are consistentwith the observation that while the smaller particles are NaF the largerparticles in high F sample are CaF2, as shown in the diffractionpattern. Finally in F+peptide containing sample, the surfacemineralization display larger, elongated particles of HApcrystallography but with random crystallographic organization (i.e.,more complete diffraction rings) while the group 6 samples displayed HApcrystallites with aligned organization, reminiscent of the HApplate-like particles within the enamel rods, again corroborating withthe SEM and EDXS results. Taken together, the most successful layerformation was accomplished using the peptide involving procedure withthe resultant mineral layer being crystalline and HAp in Group 6 andpossibly a FAp+HAp mixture in the shADP5+F case of samples, i.e., group5.

Microhardness Testing and Nanoindentation Experiments

Microhardness and nanoindentation differ in both loading range andmeasurement method as shown in the schematic in FIG. 14 and, for thesereasons, both were used in the present work to assess the mechanicaldurability of the remineralized layers on artificially formed lesions onintact human teeth. In the case of microhardness (Vickers indenter), themeasurement was a static one in that the load was applied, released andhardness was obtained by measuring the post-indentation projected areaof the indented region (foot print). This method therefore providesinformation about only the plastically deformed response (plasticdeformation) of the material. Furthermore the load applied in this studywas 10 kg, lowest allowable by the instrument. With this load, thenominal plastically deformed depth was approximately 5 μm, comparable tothe thickness of the group 6 samples (shADP5 treatment) but much deeperthan the mineralized layer of rest of the treatment groups. In addition,the actual interaction depth (long-range strain) during indentation wassignificantly deeper than 5 μm. Hence net positive contribution from theunderlying artificially formed lesion on enamel (white spot lesion, WSL)in the microhardness measurement was expected.

For groups 2-5, which mineral layers were significantly thinner than 5μm, the underlying WSL enamel was expected to have the predominanteffect on the hardness, and therefore, no statistical difference wasdetected between the group 1 (no treatment control) and groups 2-5,while slight increase in hardness was detected in group 6 compared togroup 1.

Nanoindentation, on the other hand, can handle a much smaller appliedload, e.g., in the range of 700 to 800 μN; this range of loads tomaintain a nominal total indentation depth of approximately 100 nm (<1μm in indentation width) reasonable depth for both the WSL andmineralized layer regions. The method provides a dynamic measurementwith the indentation system tracking continuously load vs. depth duringthe indentation process providing a Force-to-depth (F-d) profile of themechanical response of the sample, including biological hard tissuessuch as enamel, dentin, and bone. The hardness and elastic modulus wereextracted from the linear extrapolation of the unloading curve (see FIG.14b ). Hardness obtained by this method contains partial elasticdeformation as well as plastic deformation. With small indentation depthand footprint, indentation characterization of the mineral layer onlywas possible in cross-section for the group 6 samples where minerallayers were more than 5 μm thick. All other treatment groups, reliablenanoindentation characterization of the layers could be partiallyaccomplished because of the thin, and in some cases, discontinuousnatural of the layers. It should also be noted that the nanomechanicalproperties of the artificially created lesion were also determined, withvalues similar to those of the mineralized layers. Regardless, thecomplete nanoindentation values are tabulated in Table 10 where eachvalue represents more than 20 measurements per sample.

TABLE 10 Tabulated nanoindentation values, n ≥ 20 for each group.Elastic Test Groups Hardness (GPa) Modulus (GPa) Group 1 ( Neg Control)2.10 ± 0.26 55.1 ± 4.3 Group 2 (Ca/PO₄ Only) 2.12 ± 0.37 54.6 ± 6.3Group 3 (Low Conc. F) 2.16 ± 0.28 53.6 ± 6.2 Group 4 (High Conc. F) 2.08± 0.29 54.0 ± 6.5 Group 5 (shADF5 + Low Conc. F) 2.15 ± 0.30 55.1 ± 5.9Group 6 (shADP5) (layer) 2.23 ± 0.23 58.6 ± 4.7 (underlying enamel) 2.12± 0.21 55.0 ± 4.6 Sound Enamel  3.8 ± 0.72  91.6 ± 10.5 Sound Dentin 1.6 ± 0.33 21.8 ± 3.7

Example 10. Layer-by-Layer Mineralization by Peptide in SolutionFormulation

Summary: Using sADP peptide (SEQ ID NO:24) in aqueous solution, repeatedremineralization in separate cycles produced layered mineralization thatis fully integrated into the existing enamel structure on human tooth invitro. The implication of the results is that repeated use of dentalproducts in a variety of formulations could produce layered architectureof the remineralized structure on the surface of the teeth. Theprocedure and the products can thus be incorporated in dental healthcare products including mouth wash, whitening solutions using peptides,tooth pastes, gels and other relevant delivery systems such as dentaltrays and retainers.

Materials & Methods:

On extracted human tooth, the entire tooth was painted with lacquer(nail polish) except a 3 mm by 3 mm window on enamel. This window wassubjected to etching then remineralization. The following steps werethen carried out:

-   -   1. Etch enamel surface aqueous solution of 2.2 mM K₂HPO₄, 2.2 mM        CaCl₂, and 50 mM acetic acid at pH 4.5 for 2 weeks, changing        solution every other day, to create white spot lesion (WSL) of        pH 7.4 Tris buffer via a laboratory squeeze bottle for 30        seconds.    -   2. Soak in 100 ml of 0.8 mM sADP5 solution at 37 C for 10        minutes then blot dry with tissue paper.    -   3. Soak in 600 ml of 4.8 mM CaCl₂+2.8 mM K₂HPO₄ in pH 7.4 Tris        buffer at 37 C for 1 hour.    -   4. Drip rinse with DI water for 20 seconds then blot dry with        Kimwipe™ sheet.    -   5. Repeat steps 3-4 to add more mineralized layers.        Results & Discussions—Imaging Analysis: 4 distinctive mineral        layers of approximately 2 mm thick new mineral layers were        formed after 4 repetitions of Steps 3-5 in the mineralization        procedure.

1. Ca/P of new mineral layers is 1.43+0.15, closer to that of OCP(Stoichiometric Ca/P of OCP=1.33, HAP=1.67)

2. Ca/P of underlying enamel is close to that of HAP

Conclusions: The sound enamel and the newly formed layered mineral havethe same microstructure with well integrated interface, resulting inmechanically stable bonding. The mineralized multilayer and the soundenamel have similar chemical compositions, indicating chemicalintegration, and therefore stability.

Implications/Potential Applications:

1. Remineralize enamel surface via dental tray (also called mouth guardor retainers): user places peptide solution or other types offormulations (paste, gel, cream, varnish) in the dental tray that wouldconform and fit onto the teeth, for example, before bed or even duringthe daytime between meals (after brushing). Repeat x number ofnights/days to build up new mineral layers to achieve desired thickness.Potential treatment for hypersensitive teeth, incipient caries, whitespot lesion, and, in general preventive, daily dental health care

2. Mouth rinse, mouth wash, teeth whitening: User rinse mouth with thepeptide and mineralization solution for x time after brushing to buildup mineral layer. This procedure can be a preventative treatment orremineralization repair treatment for hypersensitive teeth, incipientcaries, white spot lesion, as well as for whitening teeth.

Example 11. Side-by-Side Comparison of Peptide (sADP5)-MineralizedWhitening Versus Commercial Whitening Products

The effect of whitening has been compared using the methods of theinvention with sADP5 (SEQ ID NO:24) to the hydrogen peroxide (HP)-basedtreatments that are frequently used in the market. These areover-the-counter and in-clinic products, i.e., a whitening strip and agel, with midlevel and high hydrogen peroxide contents, 14% and 30%,respectively. The specific commercial products used are CREST® 3DWhitening strip and ULTRADENT® OPALESCENCE®. The reasons for the choiceof these two products is as follows: The first product, whitening strip,was chosen to represent a mid-level HP content that is commerciallyavailable as OTC product for everyday use. The gel, the second product,contains very high amount of HP, which represents the clinical,chair-side whitening application. The team considered these two productsthat are available in the market as good choices for comparing withSBSI's devices, containing mineralizing peptides, sADP5, which includechair-side treatment(s) using dental tray and take-home product in thegel-form.

Procedure:

The whitening products were used according to the manufacturers'directions. For the strip, the product was applied to the pre-stainedtooth for 30 mins and peeled off. Then the samples were rinsed withwater for 1 min to clean the surface of the teeth from any residues fromthe strip. In the case of the second product, the gel was applied to thepre-stained teeth for 10 minutes and then cleaned by rinsing with waterfor 1 minute. In the peptide-remineralization case, the stained teethwere incubated with sADP5 (SEQ ID NO:24) peptide solution (0.8 mM) for10 mins., followed by soaking in the artificial saliva (for 1 hr) thatwas supplemented with 4.8 mM Ca and 2.9 mM PO₄ ionic concentrations.

Analysis & Results:

The samples treated with whitening products or with the remineralizationmethod were prepared for optical imaging for color change and also forsurface characterization with scanning electron microscopy. The imagesof the teeth samples were recorded using a light optical microscopytechnique under compact fluorescent light. The recorded images weredigitized and examined with Image-J Line (NIH Software) for quantitativeanalysis of the color change (a, d, f and h). The cross-sectionedsamples were prepared for SEM examination of the internal as well assurface structure of the teeth, and the results are shown in FIG. 15 (e,g, and i, plus their inset images).

The image in (a) shows that the tooth sample after tea staining has adarker shade, which was then used as the baseline value of the toothshade in subsequent experiments. The whitening strip increased the shadeby 5.65% while the gel treatment by 6.77%. Similar trend in whiteningimprovement of the shade was also observed in the mineralization, whichproduced 4.64% increase above the baseline value. A careful examinationof the surfaces of the teeth test samples (insets of (e) and (g))examined by SEM reveal etching of native enamel which appear as roughsurfaces because of the demineralizing effect of the HP based strip orgel products. The peptide-treated test sample, on the other hand,produces a newly formed remineralized surface (inset in image (i)).Therefore, peptide-based treatment has two simultaneous effects:whitening as well as remineralization, strengthening the enamel and,hence, resulting in healthier teeth.

The manufacturer of the commercial products cautions users of theincreased risk for tooth sensitivity and gum discomfort, both of whichare clinically attributed to enamel degradation by hydrogen peroxide(see the caution label on the right of the figure above). According toCREST® and ULTRADENT® there are no ingredients in their products thatsafeguard against enamel degradation due the action of the activeingredient, hydrogen peroxide, which while whitening the teeth, alsoadversely removes the existing mineral from the surface of the teethwith potential

In conclusion, the remineralization approach undertaken by the methodsof the invention appears to be superior to commercial products. Whilethe use HP-based products result in whitening, this is achieved at theexpense of a subtractive process in which demineralizing of the toothsurface causes loss of dental tissue with potential long-term adversedental health consequences. The peptide-based approach of the presentinvention, even in the case of solution methodology produces comparablewhitening to commercial products while also restoring the mineralstructure towards better dental health.

Example 12. Treating Dental Hypersensitivity

Summary: The materials and methods for treating dental hypersensitivityinvolve forming a mineral layer on the teeth with the enamel (crown ofthe teeth) removed exposing the dental tubules. The newly formedmineralized layer occludes exposed dentinal tubules in thispeptide-based approach forming a mechanically and thermally stablemineral structure covering the tooth damaged surface. The approachinvolves, first, creating artificial lesions by removing enamel bychemical etching to expose underlying dentin of the extracted humantooth to mimic hypersensitivity conditions. The samples were nexttreated with peptide-guided mineralization resulting in tens ofmicrometer-thick new layer over the damaged dentin. The stability of thenewly formed layer were then mechanically and thermally evaluated usingnanomechanical testing and thermal-cycling, respectively. The resultsdemonstrate that exposed dentinal tubules are successfully occluded bymechanically and thermally stable mineral layer. The methods of theinvention described herein offers a unique solution to dentinalhypersensitivity.Background: Dental hypersensitivity (DS) is one of the most commondiseases in the United States, affecting the majority adult population.The disease occurs when enamel layer, crown of the teeth, wears offexposing the underlying dentin tubules. Nerves lie at the base of thesedentinal tubules and when externally triggered, e.g., cold, hot, acid,basic conditions in the saliva, produce a sharp pain at the root of thetooth. Current approaches include desensitizing nerve ends by blockingthe axonic action via potassium salts, limiting the permeability ofdentinal tubules using synthetic adhesive sealers, e.g. NaF, bioactiveglasses, oxalic acid and glass ionomer cements, or forming coagulatesinside the tubules using protein cross-linker agents. Clinicalvalidation for these agents is still lacking and their short durabilityagainst daily tooth brushing, various foods, or drinking of acidicbeverages makes their occlusion effects incomplete.Methods: Extracted human teeth samples were collected from University ofWashington clinics. The enamel tissue was cut out the using low speedsaw on a perpendicular direction to the underlying dentin tubules. Next,the smear layer on the dentin was removed by polishing down to 0.1 umfinish and etching for 10 seconds using 10% citric acid solution. Forremineralization treatment, samples were soaked into 200 ul of 0.8 mMsADP5 peptide (SEQ ID NO:24) solution and incubated for 10 minutes at37° C. Next, excess peptide was removed by blotting and the sample wassoaked in 24 mM Tris Buffer solution supplemented with 4.8 mM Ca and2.88 mM PO₄ ions for 1 hour at 37° C. Following the remineralizationtreatment, samples were rinsed with deionized water, dried in air andcharacterized with scanning electron microscopeResults & Discussions: The teeth samples for mineralization wereproduced by removing the enamel tissue and performing a series ofpolishing and etching. As a result, as shown in FIG. 16 (first andsecond rows) dentin tubules are clearly exposed. Following the 1 hour invitro peptide remineralization treatment, these exposed dentin tubulesare successfully occluded with a newly-formed mineral which is in theform of a continuous ˜5 um layer penetrates down to tubules for ˜2-3 umdeep (FIG. 16 (third and fourth rows).Conclusion & Significance: We developed a peptide-based aqueousmineralization methodology to treat dental hypersensitivity by forming acontinuous mineral layer on the exposed dentin of human teeth in vitro.This biomimetic treatment provides a unique solution to dentinalhypersensitivity which can be used as a platform technology forin-clinic and over-the-counter hypersensitivity treatments.

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the following claims.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds, or compositions, which can ofcourse vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

Unless otherwise indicated, to the extent that definitions of terms usedherein differ between this application and references that have beenincorporated herein in their entirety, the definitions of terms usedherein presented in this application take precedence over thedefinitions presented in the references incorporated herein.

Other embodiments are set forth in the following claims.

1. A method for mineralizing teeth, comprising administering to one or more teeth of a subject in need thereof an effective amount to mineralize teeth of a polypeptide comprising or consisting of the amino acid sequence selected from the group consisting of: (shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24) (SYEKSHSQAINTDRT)₁₋₁₀ (ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP5; SEQ ID NO: 13) (PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17) (LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀; and 12-42 contiguous amino acids of (ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQ QPV(A/I)PQQPMMPVPG(H/Q)HSMTP(T/I)QH)₁₋₁₀;

or a functional equivalent thereof, or any combination thereof.
 2. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence (SYENSHSQAINVDRT)₁₋₁₀ (shADP5; SEQ ID NO:16), or a functional equivalent thereof.
 3. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence (SYEKSHSQAINTDRT)₁₋₁₀ (sADP5; SEQ ID NO:24), or a functional equivalent thereof.
 4. The method of claim 1, wherein the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: (ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4) (VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP4; SEQ ID NO: 10) (HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP8; SEQ ID NO: 21) (PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀; (ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3) (HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀; (ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11) (HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀; (ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHHLPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀; (ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀; (ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23) (PAQQPVIPQQPM1VIP)₁₋₁₀; (ADP3M; SEQ ID NO: 8) (WPATDKTKREEVD)₁₋₁₀; and (ADP3H; SEQ ID NO: 9) (WPSTDKTKREEVD)₁₋₁₀,

or a functional equivalent thereof, or a combination thereof.
 5. The method of claim 1, wherein the polypeptide comprises a fusion polypeptide comprising two or more of the amino acid sequences selected from the group consisting of: (shADP5; SEQ ID NO: 16) (SYENSHSQAINVDRT)₁₋₁₀; (sADP5; SEQ ID NO: 24) (SYEKSHSQAINTDRT)₁₋₁₀ (ADP1; SEQ ID NO: 1) (HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP2; SEQ ID NO: 4) (VPG(H/Q)HSMTP(T/I)QH)₁₋₁₀; (ADP3; SEQ ID NO: 7) (WP(A/S)TDKTKREEVD)₁₋₁₀; (ADP4; SEQ ID NO: 10) (HPP(S/T)HTLQPHHH(L/I)PVV)₁₋₁₀; (ADP5; SEQ ID NO: 13) (PGYIN(L/F)SYE(K/N)SHSQAIN(T/V)DRTA)₁₋₁₀; (ADP6; SEQ ID NO: 17) (LPPLFSMPLSPILPELPLEAWPAT)₁₋₁₀; (ADP8; SEQ ID NO: 21) (PAQQPV(A/I)PQQPMMP)₁₋₁₀; (ADP7; SEQ ID NO: 18) (HPP(S/T)HTLQPHHH(L/I)PVVPAQQPV(A/I)PQQPMMPVPG(H/Q) HSMTP(T/I)QH)₁₋₁₀; (ADP1M; SEQ ID NO: 2) HTLQPHHHLPVV)₁₋₁₀; (ADP1H; SEQ ID NO: 3) (HTLQPHHHIPVV)₁₋₁₀; (ADP2M; SEQ ID NO: 5) (VPGHHSMTPTQH)₁₋₁₀; (ADP2H; SEQ ID NO: 6) (VPGQHSMTPIQH)₁₋₁₀; (ADP4M; SEQ ID NO: 11) (HPPSHTLQPHHHLPVV)₁₋₁₀; (ADP4H; SEQ ID NO: 12) (HPPTHTLQPHHHIPVV)₁₋₁₀; (ADP7M; SEQ ID NO: 19) (HPPSHTLQPHHELPVVPAQQPVAPQQPMMPVPGHHSMTPTQH)₁₋₁₀; (ADP7H; SEQ ID NO: 20) (HPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQH)₁₋₁₀; (ADP8M; SEQ ID NO: 22) (PAQQPVAPQQPMMP)₁₋₁₀; and (ADP8H; SEQ ID NO: 23) (PAQQPVIPQQPMMP)₁₋₁₀;

or a functional equivalent thereof.
 6. The method of claim 1, wherein the polypeptide is present in a single copy.
 7. The method of claim 1, further comprising administering to the subject at least one calcium ion source and at least one phosphate ion source.
 8. The method of claim 7 wherein the calcium ion source is selected from the group consisting of calcium acetate, calcium carbonate, calcium citrate, calcium chloride, calcium gluconate, calcium glycerophosphate, calcium lactate, and calcium phosphate.
 9. The method of claim 7, wherein the phosphate ion source is selected from the group consisting of aluminum phosphates, calcium phosphates, potassium phosphates, and sodium phosphates.
 10. The method of claim 7, wherein the method comprising administering a formulation of the polypeptide, at least one calcium ion source and at least one phosphate ion source, wherein the formulation has (a) a Ca²⁺ concentration ranging between about 1 mM and about 2 M, between about 1 mM and about 1M, between about 1 mM and about 0.5M, between about 1 mM and about 100 mM, between about 1 mM and about 50 mM, between about 1 mM and about 10 mM, between about 2 mM and about 8 mM, between about 3 mM and about 7 mM, between about 4 mM and about 6 mM, or between about 4.5 mM and about 5.5 mM; and (b) a PO₄ ³⁻ concentration ranging between about 0.5 mM and about 2 M, between about 0.5 mM and about 1M, between about 0.5 mM and about 0.5M, between about 0.5 mM and about 100 mM, between about 0.5 mM and about 50 mM, between about 0.5 mM and about 10 mM, between about 0.5 mM and about 7 mM, or between about 1 mM and about 6 mM, between about 1.5 mM and about 5 mM, between about 2 mM and about 4 mM, or between about 2.5 mM and about 3.5 mM.
 11. The method of claim 1, wherein the polypeptide is detectably labeled.
 12. The method of claim 1, wherein the method further comprises administering fluoride to the subject.
 13. The method of claim 1, wherein the formulation further comprises fluoride.
 14. The method of claim 13, wherein the fluoride is present in the formulation at between about between about 50 parts per million (ppm) and about 20,000 ppm, between about 50 ppm and about 10,000 ppm, between about 50 ppm and about 5000 ppm, between about 50 ppm and about 1000 ppm, between about 50 ppm and about 500 ppm, or about 75 ppm and about 400 ppm, between about 100 ppm and about 300 ppm, between about 150 ppm and about 250 ppm, or about 200 ppm.
 15. The method of claim 10, wherein the formulation is selected from the group consisting of toothpaste, toothpowders, mouthwash, gel, dental floss, liquid dentifrices, dental tablets, topical gels, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, lozenges, tooth trays, tooth varnishes, and food products.
 16. The method of claim 10, wherein the formulation comprises a lozenge, a gel, or mouthwash.
 17. The method of claim 1 wherein the polypeptide is administered in a formulation at a concentration of between about 0.01 mM and about 0.5M, between about 0.01 mM and about 0.1M, between about 0.01 mM and about 50 mM, between about 0.01 mM to about 20 mM, or between about 0.1 mM to about 15 mM, or between about 0.5 mM to about 12.5 mM, or between about 0.8 mM and about 10 mM.
 18. The method of claim 1, wherein the subject is one suffering from demineralization of enamel of one or more tooth. 19-21. (canceled)
 22. A polypeptide consisting of the amino acid sequence of (SYENSHSQAINVDRT)₁₋₁₀ (shADP5; SEQ ID NO:16); or (SYEKSHSQAINTDRT)₁₋₁₀ (sADP5; SEQ ID NO:24).
 23. (canceled)
 24. An oral care product, comprising (a) the polypeptide of claim 22, (b) at least one calcium ion source, and (c) at least one phosphate ion source. 25-34. (canceled) 